Antisense modulation of PTP1B expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of PTP1B. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PTP1B. Methods of using these compounds for modulation of PTP1B expression and for treatment of diseases associated with expression of PTP1B are provided.

BACKGROUND OF THE INVENTION

[0001] The process of phosphorylation, defined as the attachment of aphosphate moiety to a biological molecule through the action of enzymescalled kinases, represents one course by which intracellular signals arepropagated resulting finally in a cellular response. Within the cell,proteins can be phosphorylated on serine, threonine or tyrosine residuesand the extent of phosphorylation is regulated by the opposing action ofphosphatases, which remove the phosphate moieties. While the majority ofprotein phosphorylation within the cell is on serine and threonineresidues, tyrosine phosphorylation is modulated to the greatest extentduring oncogenic transformation and growth factor stimulation (Zhang,Crit. Rev. Biochem. Mol. Biol., 1998, 33, 1-52) .

[0002] Because phosphorylation is such a ubiquitous process within cellsand because cellular phenotypes are largely influenced by the activityof these pathways, it is currently believed that a number of diseasestates and/or disorders are a result of either aberrant activation of,or functional mutations in, kinases and phosphatases. Consequently,considerable attention has been devoted recently to the characterizationof tyrosine kinases and tyrosine phosphatases.

[0003] PTP1B (also known as protein phosphatase 1B and PTPN1) is anendoplasmic reticulum (ER)-associated enzyme originally isolated as themajor protein tyrosine phosphatase of the human placenta (Tonks et al.,J. Biol. Chem., 1988, 263, 6731-6737; Tonks et al., J. Biol. Chem.,1988, 263, 6722-6730).

[0004] An essential regulatory role in signaling mediated by the insulinreceptor has been established for PTP1B. PTP1B interacts with anddephosphorylates the activated insulin receptor both in vitro and inintact cells resulting in the downregulation of the signaling pathway(Goldstein et al., Mol. Cell. Biochem., 1998, 182, 91-99; Seely et al.,Diabetes, 1996, 45, 1379-1385). In addition, PTP1B modulates themitogenic actions of insulin (Goldstein et al., Mol. Cell. Biochem.,1998, 182, 91-99). In rat adipose cells overexpressing PTP1B, thetranslocation of the GLUT4 glucose transporter was inhibited,implicating PTP1B as a negative regulator of glucose transport as well(Chen et al., J. Biol. Chem., 1997, 272, 8026-8031).

[0005] Mouse knockout models lacking the PTP1B gene also point towardthe negative regulation of insulin signaling by PTP1B. Mice harboring adisrupted PTP1B gene showed increased insulin sensitivity, increasedphosphorylation of the insulin receptor and when placed on a high-fatdiet, PTP1B −/− mice were resistant to weight gain and remained insulinsensitive (Elchebly et al., Science, 1999, 283, 1544-1548). Thesestudies clearly establish PTP1B as a therapeutic target in the treatmentof diabetes and obesity.

[0006] PTP1B, which is differentially regulated during the cell cycle(Schievella et al., Cell. Growth Differ., 1993, 4, 239-246), isexpressed in insulin sensitive tissues as two different isoforms thatarise from alternate splicing of the pre-mRNA (Shifrin and Neel, J.Biol. Chem., 1993, 268, 25376-25384). It was recently demonstrated thatthe ratio of the alternatively spliced products is affected by growthfactors such as insulin and differs in various tissues examined (Selland Reese, Mol. Genet. Metab., 1999, 66, 189-192). In these studies itwas also found that the levels of the variants correlated with theplasma insulin concentration and percentage body fat and may thereforebe used as a biomarker for patients with chronic hyperinsulinemia ortype 2 diabetes.

[0007] Liu and Chernoff have shown that PTP1B binds to and serves as asubstrate for the epidermal growth factor receptor (EGFR) (Liu andChernoff, Biochem. J., 1997, 327, 139-145). Furthermore, in A431 humanepidermoid carcinoma cells, PT1B was found to be inactivated by thepresence of H₂O₂ generated by the addition of EGF. These studiesindicate that PTP1B can be negatively regulated by the oxidation stateof the cell, which is often deregulated during tumorigenesis (Lee etal., J. Biol. Chem., 1998, 273, 15366-15372).

[0008] Overexpression of PTP1B has been demonstrated in malignantovarian cancers and this correlation was accompanied by a concomitantincrease in the expression of the associated growth factor receptor(Wiener et al., Am. J. Obstet. Gynecol., 1994, 170, 1177-1183).

[0009] PTP1B has been shown to suppress transformation in NIH3T3 cellsinduced by the neu oncogene (Brown-Shimer et al., Cancer Res., 1992, 52,478-482), as well as in rat 3Y1 fibroblasts induced by v-srk, v-src, andv-ras (Liu et al., Mol. Cell. Biol., 1998, 18, 250-259) and rat-1fibroblasts induced by bcr-abl (LaMontagne et al., Proc. Natl. Acad.Sci. U.S.A., 1998, 95, 14094-14099). It has also been shown that PTP1Bpromotes differentiation of K562 cells, a chronic myelogenous leukemiacell line, in a similar manner as does an inhibitor of the bcr-abloncoprotein. These studies describe the possible role of PTP1B incontrolling the pathogenesis of chronic myeloid leukemia (LaMontagne etal., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 14094-14099).

[0010] PTP1B negatively regulates integrin signaling by interacting withone or more adhesion-dependent signaling components and repressingintegrin-mediated MAP kinase activation (Liu et al., Curr. Biol., 1998,8, 173-176). Other studies designed to study integrin signaling, using acatalytically inactive form of PTP1B, have shown that PTP1B regulatescadherin-mediated cell adhesion (Balsamo et al., J. Cell. Biol., 1998,143, 523-532) as well as cell spreading, focal adhesion and stress fiberformation and tyrosine phosphorylation (Arregui et al., J. Cell. Biol.,1998, 143, 861-873).

[0011] Currently, therapeutic agents designed to inhibit the synthesisor action of PTP1B include small molecules (Ham et al., Bioorg. Med.Chem. Lett., 1999, 9, 185-186; Skorey et al., J. Biol. Chem., 1997, 272,22472-22480; Taing et al., Biochemistry, 1999, 38, 3793-3803; Taylor etal., Bioorg. Med. Chem., 1998, 6, 1457-1468; Wang et al., Bioorg. Med.Chem. Lett., 1998, 8, 345-350; Wang et al., Biochem. Pharmacol., 1997,54, 703-711; Yao et al., Bioorg. Med. Chem., 1998, 6, 1799-1810) andpeptides (Chen et al., Biochemistry, 1999, 38, 384-389; Desmarais etal., Arch. Biochem. Biophys., 1998, 354, 225-231; Roller et al., Bioorg.Med. Chem. Lett., 1998, 8, 2149-2150). In addition, disclosed in the PCTpublication WO 97/32595 are phosphopeptides and antibodies that inhibitthe association of PTP1B with the activated insulin receptor for thetreatment of disorders associated with insulin resistance. Antisensenucleotides against PTP1B are also generally disclosed (Olefsky, 1997).

[0012] There remains a long felt need for additional agents capable ofeffectively inhibiting PTP1B function and antisense technology isemerging as an effective means for reducing the expression of specificgene products. This technology may therefore prove to be uniquely usefulin a number of therapeutic, diagnostic, and research applications forthe modulation of PTP1B expression.

[0013] The present invention, therefore, provides compositions andmethods for modulating PTP1B expression, including modulation of thealternatively spliced form of PTP1B.

SUMMARY OF THE INVENTION

[0014] The present invention provides compositions and methods formodulating the expression of PTP1B. In particular, this inventionrelates to compounds, particularly antisense oligonucleotides,specifically hybridizable with nucleic acids encoding PTP1B. Sucholigonucleotides have been shown to modulate the expression of PTP1B.

[0015] The present invention is directed to antisense compounds,particularly oligonucleotides, which are targeted to a nucleic acidencoding PTP1B, and which modulate the expression of PTP1B.Pharmaceutical and other compositions comprising the antisense compoundsof the invention are also provided. Further provided are methods ofmodulating the expression of PTP1B in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofPTP1B by administering a therapeutically or prophylactically effectiveamount of one or more of the antisense compounds or compositions of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding PTP1B, ultimately modulating the amountof PTP1B produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingPTP1B. As used herein, the terms “target nucleic acid” and “nucleic acidencoding PTP1B” encompass DNA encoding PTP1B, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”.

[0017] The functions of DNA to be interfered with include replicationand transcription. The functions of RNA to be interfered with includeall vital functions such as, for example, translocation of the RNA tothe site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of PTP1B. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

[0018] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding PTP1B. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding PTP1B, regardless of the sequence(s) of such codons.

[0019] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0020] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0021] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0022] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0023] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementary orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0024] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0025] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotides have been safely and effectively administered to humansand numerous clinical trials are presently underway. It is thusestablished that oligonucleotides can be useful therapeutic modalitiesthat can be configured to be useful in treatment regimes for treatmentof cells, tissues and animals, especially humans. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0026] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0027] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0028] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0029] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0030] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0031] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0032] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0033] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— ((knownas a methylene (methylimino) or MMI backbone)), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂ (wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—)of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0034] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.

[0035] A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0036] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5, 646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

[0037] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0038] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and U.S. Pat. No. 5,750,692, which is commonly owned withthe instant application and also herein incorporated by reference.

[0039] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0040] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0041] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0042] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0043] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0044] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0045] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0046] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0047] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

[0048] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0049] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoc acid, naphthalene-2-sulfonicacid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0050] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0051] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of PTP1B is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0052] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding PTP1B, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding PTP1B canbe detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of PTP1B in a sample may also beprepared.

[0053] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0054] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0055] Compositions and formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0056] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0057] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0058] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0059] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0060] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

[0061] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0062] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0063] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0064] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0065] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0066] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0067] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0068] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0069] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0070] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0071] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0072] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0073] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

[0074] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0075] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0076] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0077] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0078] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0079] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0080] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0081] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0082] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0083] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0084] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0085] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising NovasomeJ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeJ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0086] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideGM_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1), or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0087] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

[0088] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxy-nucleotides in liposomes. WO 97/04787 toLove et al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0089] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0090] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0091] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0092] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0093] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0094] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0095] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0096] Penetration Enhancers:

[0097] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0098] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0099] Surfactants:

[0100] In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

[0101] Fatty Acids:

[0102] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0103] Bile Salts:

[0104] The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0105] Chelating Agents:

[0106] Chelating agents, as used in connection with the presentinvention, can be defined as compounds that remove metallic ions fromsolution by forming complexes therewith, with the result that absorptionof oligonucleotides through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0107] Non-chelating Non-surfactants:

[0108] As used herein, non-chelating non-surfactant penetrationenhancing compounds can be defined as compounds that demonstrateinsignificant activity as chelating agents or as surfactants but thatnonetheless enhance absorption of oliqonucleotides through thealimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

[0109] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0110] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0111] Carriers:

[0112] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0113] Excipients:

[0114] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0115] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0116] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0117] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0118] Other Components:

[0119] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0120] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0121] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemo-therapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0122] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0123] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0124] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

[0125] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham, Mass. or Glen Research, Inc., Sterling, Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0126] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling, Va., or ChemGenes,Needham, Mass.).

2′-Fluoro Amidites 2′-Fluorodeoxyadenosine Amidites

[0127] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyi groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

[0128] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

[0129] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

[0130] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

[0131] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0132] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

[0133] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-16° C.,the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0134] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0135] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0136] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0137] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0138] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0139]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxytetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0140] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0141] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0142] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase. The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0143]5′-O-tert-Butyidiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0144]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0145]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for. 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0146] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂) . Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0147] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

540-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0148] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) Nucleoside Amidites

[0149] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0150] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0151] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0152] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethyl aminoethoxy)ethyl)]-5-methylUridine

[0153] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0154] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

[0155] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0156] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0157] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0158] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0159] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0160] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0161] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0162] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0163] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside Synthesis

[0164] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0165] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0166] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0167] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0168] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0169] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0170] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0171] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0172] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0173] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0174] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0175] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

[0176] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0177] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 5 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

T-24 Cells

[0178] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0179] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

A549 Cells

[0180] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells

[0181] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

HEK Cells

[0182] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

PC-12 Cells

[0183] The rat neuronal cell line PC-12 was obtained from the AmericanType Culure Collection (Manassas, Va.). PC-12 cells were routinelycultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg,Md.) supplemented with 10% horse serum+5% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 20000cells/well for use in RT-PCR analysis.

[0184] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds

[0185] When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0186] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of PTP1B Expression

[0187] Antisense modulation of PTP1B expression can be assayed in avariety of ways known in the art. For example, PTP1B mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Prior to quantitative PCRanalysis, primer-probe sets specific to the target gene being measuredare evaluated for their ability to be “multiplexed” with a GAPDHamplification reaction. In multiplexing, both the target gene and theinternal standard gene GAPDH are amplified concurrently in a singlesample. In this analysis, mRNA isolated from untreated cells is seriallydiluted. Each dilution is amplified in the presence of primer-probe setsspecific for GAPDH only, target gene only (“single-plexing”), or both(multiplexing). Following PCR amplification, standard curves of GAPDHand target mRNA signal as a function of dilution are generated from boththe single-plexed and multiplexed samples. If both the slope andcorrelation coefficient of the GAPDH and target signals generated fromthe multiplexed samples fall within 10% of their corresponding valuesgenerated from the single-plexed samples, the primer-probe set specificfor that target is deemed as multiplexable. Other methods of PCR arealso known in the art.

[0188] Protein levels of PTP1B can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to PTP1B can be identified and obtained froma variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0189] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

[0190] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0191] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0192] Total mRNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following the manufacturers recommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

[0193] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-time Quantitative PCR Analysis of PTP1B mRNA Levels

[0194] Quantitation of PTP1B mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMPA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0195] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

[0196] Probes and primers to human PTP1B were designed to hybridize to ahuman PTP1B sequence, using published sequence information (GenBankaccession number M31724, incorporated herein as SEQ ID NO:3). For humanPTP1B the PCR primers were:

[0197] forward primer: GGAGTTCGAGCAGATCGACAA (SEQ ID NO: 4)

[0198] reverse primer: GGCCACTCTACATGGGAAGTC (SEQ ID NO: 5) and

[0199] the PCR probe was: FAM-AGCTGGGCGGCCATTTACCAGGAT-TAMRA (SEQ ID NO:6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For human GAPDH the PCR primers were:

[0200] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

[0201] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and

[0202] the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ IDNO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMPA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

[0203] Probes and primers to rat PTP1B were designed to hybridize to arat PTP1B sequence, using published sequence information (GenBankaccession number M33962, incorporated herein as SEQ ID NO:10). For ratPTP1B the PCR primers were:

[0204] forward primer: CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:11)

[0205] reverse primer: CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12) and

[0206] the PCR probe was: FAM-CGACTCGTCAGTGCAGGATCAGTGGA-TAMRA (SEQ IDNO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For rat GAPDH the PCR primers were:

[0207] forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 14)

[0208] reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 15) and

[0209] the PCR probe was: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMPA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14 Northern Blot Analysis of PTP1B mRNA Levels

[0210] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0211] To detect human PTP1B, a human PTP1B specific probe was preparedby PCR using the forward primer GGAGTTCGAGCAGATCGACAA (SEQ ID NO: 4) andthe reverse primer GGCCACTCTACATGGGAAGTC (SEQ ID NO: 5). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0212] To detect rat PTP1B, a rat PTP1B specific probe was prepared byPCR using the forward primer CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:11) andthe reverse primer CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for rat glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0213] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human PTP1B Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0214] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanPTP1B RNA, using published sequences (GenBank accession number M31724,incorporated herein as SEQ ID NO: 3). The oligonucleotides are shown inTable 1. ATarget siteA indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human PTP1B mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibitionof human PTP1B mRNA levels by chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap ISIS TARGET TARGET # REGION SEQ IDNO SITE SEQUENCE % INHIB SEQ ID NO 107769 5′ UTR 3 1cttagccccgaggcccgccc 0 17 107770 5′ UTR 3 41 ctcggcccactgcgccgtct 58 18107771 Start 3 74 catgacgggccagggcggct 60 19 Codon 107772 Coding 3 113cccggacttgtcgatctgct 95 20 107773 Coding 3 154 ctggcttcatgtcggatatc 8821 107774 Coding 3 178 ttggccactctacatgggaa 77 22 107775 Coding 3 223ggactgacgtctctgtacct 75 23 107776 Coding 3 252 gatgtagtttaatccgacta 8224 107777 Coding 3 280 ctagcgttgatatagtcatt 29 25 107778 Coding 3 324gggtaagaatgtaactcctt 86 26 107779 Coding 3 352 tgaccgcatgtgttaggcaa 7527 107780 Coding 3 381 ttttctgctcccacaccatc 30 28 107781 Coding 3 408ctctgttgagcatgacgaca 78 29 107782 Coding 3 436 gcgcattttaacgaaccttt 8330 107783 Coding 3 490 aaatttgtgtcttcaaagat 0 31 107784 Coding 3 519tgatatcttcagagatcaat 57 32 107785 Coding 3 547 tctagctgtcgcactgtata 7433 107786 Coding 3 575 agtttcttgggttgtaaggt 33 34 107787 Coding 3 604gtggtatagtggaaatgtaa 51 35 107788 Coding 3 632 tgattcagggactccaaagt 5536 107789 Coding 3 661 ttgaaaagaaagttcaagaa 17 37 107790 Coding 3 688gggctgagtgaccctgactc 61 38 107791 Coding 3 716 gcagtgcaccacaacgggcc 8139 107792 Coding 3 744 aggttccagacctgccgatg 81 40 107793 Coding 3 772agcaggaggcaggtatcagc 2 41 107794 Coding 3 799 gaagaagggtctttcctctt 53 42107795 Coding 3 826 tctaacagcactttcttgat 18 43 107796 Coding 3 853atcaaccccatccgaaactt 0 44 107797 Coding 3 880 gagaagcgcagctggtcggc 82 45107798 Coding 3 908 tttggcaccttcgatcacag 62 46 107799 Coding 3 952agctccttccactgatcctg 70 47 107800 Coding 3 1024 tccaggattcgtttgggtgg 7248 107801 Coding 3 1052 gaactccctgcatttcccat 68 49 107802 Coding 3 1079ttccttcacccactggtgat 40 50 107803 Coding 3 1148 gtagggtgcggcatttaagg 051 107804 Coding 3 1176 cagtgtcttgactcatgctt 75 52 107805 Coding 3 1222gcctgggcacctcgaagact 67 53 107806 Coding 3 1268 ctcgtccttctcgggcagtg 3754 107807 Coding 3 1295 gggcttccagtaactcagtg 73 55 107808 Coding 3 1323ccgtagccacgcacatgttg 80 56 107809 Coding 3 1351 tagcagaggtaagcgccggc 7257 107810 Stop 3 1379 ctatgtgttgctgttgaaca 85 58 Codon 107811 3′ UTR 31404 ggaggtggagtggaggaggg 51 59 107812 3′ UTR 3 1433ggctctgcgggcagaggcgg 81 60 107813 3′ UTR 3 1460 ccgcggcatgcctgctagtc 8461 107814 3′ UTR 3 1489 tctctacgcggtccggcggc 84 62 107815 3′ UTR 3 1533aagatgggttttagtgcaga 65 63 107816 3′ UTR 3 1634 gtactctctttcactctcct 6964 107817 3′ UTR 3 1662 ggccccttccctctgcgccg 59 65 107818 3′ UTR 3 1707ctccaggagggagccctggg 57 66 107819 3′ UTR 3 1735 gggctgttggcgtgcgccgc 5467 107820 3′ UTR 3 1783 tttaaataaatatggagtgg 0 68 107821 3′ UTR 3 1831gttcaagaaaatgctagtgc 69 69 107822 3′ UTR 3 1884 ttgataaagcccttgatgca 7470 107823 3′ UTR 3 1936 atggcaaagccttccattcc 26 71 107824 3′ UTR 3 1973gtcctccttcccagtactgg 60 72 107825 3′ UTR 3 2011 ttacccacaatatcactaaa 3973 107826 3′ UTR 3 2045 attatatattatagcattgt 24 74 107827 3′ UTR 3 2080tcacatcatgtttcttatta 48 75 107828 3′ UTR 3 2115 ataacagggaggagaataag 076 107829 3′ UTR 3 2170 ttacatgcattctaatacac 21 77 107830 3′ UTR 3 2223gatcaaagtttctcatttca 81 78 107831 3′ UTR 3 2274 ggtcatgcacaggcaggttg 8279 107832 3′ UTR 3 2309 caacaggcttaggaaccaca 65 80 107833 3′ UTR 3 2344aactgcaccctattgctgag 61 81 107834 3′ UTR 3 2380 gtcatgccaggaattagcaa 082 107835 3′ UTR 3 2413 acaggctgggcctcaccagg 58 83 107836 3′ UTR 3 2443tgagttacagcaagaccctg 44 84 107837 3′ UTR 3 2473 gaatatggcttcccataccc 085 107838 3′ UTR 3 2502 ccctaaatcatgtccagagc 87 86 107839 3′ UTR 3 2558gacttggaatggcggaggct 74 87 107840 3′ UTR 3 2587 caaatcacggtctgctcaag 3188 107841 3′ UTR 3 2618 gaagtgtggtttccagcagg 56 89 107842 3′ UTR 3 2648cctaaaggaccgtcacccag 42 90 107843 3′ UTR 3 2678 gtgaaccgggacagagacgg 2591 107844 3′ UTR 3 2724 gccccacagggtttgagggt 53 92 107845 3′ UTR 3 2755cctttgcaggaagagtcgtg 75 93 107846 3′ UTR 3 2785 aaagccacttaatgtggagg 7994 107847 3′ UTR 3 2844 gtgaaaatgctggcaagaga 86 95 107848 3′ UTR 3 2970tcagaatgcttacagcctgg 61 96

[0215] As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 26,27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48, 49, 50, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 72,73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 94, 95, and 96demonstrated at least 35% inhibition of human PTP1B expression in thisassay and are therefore preferred.

Example 16 Antisense Inhibition of Rat PTP1B Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0216] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the ratPTP1B RNA, using published sequences (GenBank accession number M33962,incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown inTable 2. ATarget siteA indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on rat PTP1B mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”. TABLE 2 Inhibitionof rat PTP1B mRNA levels by chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap ISIS TARGET TARGET # REGION SEQ IDNO SITE SEQUENCE % INHIB SEQ ID NO 111549 5′ UTR 10 1caacctccccagcagcggct 32 97 111550 5′ UTR 10 33 tcgaggcccgtcgcccgcca 2798 111551 5′ UTR 10 73 cctcggccgtccgccgcgct 34 99 111552 Coding 10 132tcgatctgctcgaattcctt 49 100 113669 Coding 10 164 cctggtaaatagccgcccag 36101 113670 Coding 10 174 tgtcgaatatcctggtaaat 63 102 113671 Coding 10184 actggcttcatgtcgaatat 58 103 113672 Coding 10 189aagtcactggcttcatgtcg 40 104 111553 Coding 10 190 gaagtcactggcttcatgtc 27105 113673 Coding 10 191 ggaagtcactggcttcatgt 54 106 113674 Coding 10192 gggaagtcactggcttcatg 41 107 113675 Coding 10 193tgggaagtcactggcttcat 56 108 113676 Coding 10 194 atgggaagtcactggcttca 31109 113677 Coding 10 195 catgggaagtcactggcttc 59 110 113678 Coding 10225 tttttgttcttaggaagttt 24 111 111554 Coding 10 228cggtttttgttcttaggaag 45 112 111555 Coding 10 269 tccgactgtggtcaaaaggg 39113 113679 Coding 10 273 ttaatccgactgtggtcaaa 45 114 113680 Coding 10298 atagtcattatcttcctgat 49 115 111556 Coding 10 303ttgatatagtcattatcttc 29 116 113681 Coding 10 330 gcttcctccatttttatcaa 67117 111557 Coding 10 359 ggccctgggtgaggatatag 20 118 113682 Coding 10399 cacaccatctcccagaagtg 29 119 111558 Coding 10 405tgctcccacaccatctccca 48 120 113683 Coding 10 406 ctgctcccacaccatctccc 51121 113684 Coding 10 407 tctgctcccacaccatctcc 37 122 113685 Coding 10408 ttctgctcccacaccatctc 54 123 113686 Coding 10 417cccctgctcttctgctccca 60 124 111559 Coding 10 438 atgcggttgagcatgaccac 15125 113687 Coding 10 459 tttaacgagcctttctccat 33 126 113688 Coding 10492 ttttcttctttctgtggcca 54 127 113689 Coding 10 502gaccatctctttttcttctt 58 128 111560 Coding 10 540 tcagagatcagtgtcagctt 21129 113690 Coding 10 550 cttgacatcttcagagatca 64 130 113691 Coding 10558 taatatgacttgacatcttc 46 131 111561 Coding 10 579aactccaactgccgtactgt 14 132 111562 Coding 10 611 tctctcgagcctcctgggta 38133 113692 Coding 10 648 ccaaagtcaggccaggtggt 63 134 111563 Coding 10654 gggactccaaagtcaggcca 31 135 113693 Coding 10 655agggactccaaagtcaggcc 50 136 113694 Coding 10 656 cagggactccaaagtcaggc 45137 113695 Coding 10 657 tcagggactccaaagtcagg 49 138 113696 Coding 10663 ggtgactcagggactccaaa 34 139 111564 Coding 10 705cctgactctcggactttgaa 53 140 113697 Coding 10 715 gctgagtgagcctgactctc 57141 113698 Coding 10 726 ccgtgctctgggctgagtga 48 142 111565 Coding 10774 aaggtccctgacctgccaat 28 143 111566 Coding 10 819tctttcctcttgtccatcag 34 144 113699 Coding 10 820 gtctttcctcttgtccatca 41145 113700 Coding 10 821 ggtctttcctcttgtccatc 66 146 113701 Coding 10822 gggtctttcctcttgtccat 71 147 113702 Coding 10 852aacagcactttcttgatgtc 39 148 111567 Coding 10 869 ggaacctgcgcatctccaac 0149 111568 Coding 10 897 tggtcggccgtctggatgag 29 150 113703 Coding 10909 gagaagcgcagttggtcggc 48 151 113704 Coding 10 915aggtaggagaagcgcagttg 31 152 113705 Coding 10 918 gccaggtaggagaagcgcag 41153 111569 Coding 10 919 agccaggtaggagaagcgca 56 154 113706 Coding 10920 cagccaggtaggagaagcgc 58 155 113707 Coding 10 921acagccaggtaggagaagcg 43 156 113708 Coding 10 922 cacagccaggtaggagaagc 49157 113709 Coding 10 923 tcacagccaggtaggagaag 47 158 111570 Coding 10924 atcacagccaggtaggagaa 51 159 113710 Coding 10 925gatcacagccaggtaggaga 51 160 113711 Coding 10 926 cgatcacagccaggtaggag 63161 113712 Coding 10 927 tcgatcacagccaggtagga 71 162 113713 Coding 10932 caccctcgatcacagccagg 75 163 113714 Coding 10 978tccttccactgatcctgcac 97 164 111571 Coding 10 979 ctccttccactgatcctgca 89165 113715 Coding 10 980 gctccttccactgatcctgc 99 166 107799 Coding 10981 agctccttccactgatcctg 99 167 113716 Coding 10 982aagctccttccactgatcct 97 168 113717 Coding 10 983 aaagctccttccactgatcc 95169 113718 Coding 10 984 gaaagctccttccactgatc 95 170 113719 Coding 10985 ggaaagctccttccactgat 95 171 111572 Coding 10 986gggaaagctccttccactga 89 172 113720 Coding 10 987 tgggaaagctccttccactg 97173 113721 Coding 10 1036 tggccggggaggtgggggca 20 174 111573 Coding 101040 tgggtggccggggaggtggg 20 175 113722 Coding 10 1046tgcgtttgggtggccgggga 18 176 111574 Coding 10 1073 tgcacttgccattgtgaggc38 177 113723 Coding 10 1206 acttcagtgtcttgactcat 67 178 113724 Coding10 1207 aacttcagtgtcttgactca 60 179 111575 Coding 10 1208taacttcagtgtcttgactc 50 180 113725 Coding 10 1209 ctaacttcagtgtcttgact53 181 111576 Coding 10 1255 gacagatgcctgagcacttt 32 182 106409 Coding10 1333 gaccaggaagggcttccagt 32 183 113726 Coding 10 1334tgaccaggaagggcttccag 39 184 111577 Coding 10 1335 ttgaccaggaagggcttcca32 185 113727 Coding 10 1336 gttgaccaggaagggcttcc 41 186 113728 Coding10 1342 gcacacgttgaccaggaagg 59 187 111578 Coding 10 1375gaggtacgcgccagtcgcca 45 188 111579 Coding 10 1387 tacccggtaacagaggtacg32 189 111580 Coding 10 1397 agtgaaaacatacccggtaa 30 190 111581 3′ UTR10 1456 caaatcctaacctgggcagt 31 191 111582 3′ UTR 10 1519ttccagttccaccacaggct 24 192 111583 3′ UTR 10 1552 ccagtgcacagatgcccctc47 193 111584 3′ UTR 10 1609 acaggttaaggccctgagat 29 194 111585 3′ UTR10 1783 gcctagcatcttttgttttc 43 195 111586 3′ UTR 10 1890aagccagcaggaactttaca 36 196 111587 3′ UTR 10 2002 gggacacctgagggaagcag16 197 111588 3′ UTR 10 2048 ggtcatctgcaagatggcgg 40 198 111589 3′ UTR10 2118 gccaacctctgatgaccctg 25 199 111590 3′ UTR 10 2143tggaagccccagctctaagc 25 200 111591 3′ UTR 10 2165 tagtaatgactttccaatca44 201 111592 3′ UTR 10 2208 tgagtcttgctttacacctc 41 202 111593 3′ UTR10 2252 cctgcgcgcggagtgacttc 22 203 111594 3′ UTR 10 2299aggacgtcactgcagcagga 43 204 111595 3′ UTR 10 2346 tcaggacaagtcttggcagt32 205 111596 3′ UTR 10 2405 gaggctgcacagtaagcgct 34 206 111597 3′ UTR10 2422 tcagccaaccagcatcagag 20 207 111598 3′ UTR 10 2449acccacagtgtccacctccc 30 208 111599 3′ UTR 10 2502 agtgcgggctgtgctgctgg30 209 111600 3′ UTR 10 2553 cagctcgctctggcggcctc 8 210 111601 3′ UTR 102608 aggaagggagctgcacgtcc 32 211 111602 3′ UTR 10 2664ccctcacgattgctcgtggg 24 212 111603 3′ UTR 10 2756 cagtggagcggctcctctgg18 213 111604 3′ UTR 10 2830 caggctgacaccttacacgg 30 214 111605 3′ UTR10 2883 gtcctacctcaaccctagga 37 215 111606 3′ UTR 10 2917ctgccccagcaccagccaca 12 216 111607 3′ UTR 10 2946 attgcttctaagaccctcag33 217 111608 3′ UTR 10 2978 ttacatgtcaccactgttgt 28 218 111609 3′ UTR10 3007 tacacatgtcatcagtagcc 37 219 111610 3′ UTR 10 3080ttttctaactcacagggaaa 30 220 111611 3′ UTR 10 3153 gtgcccgccagtgagcaggc23 221 111612 3′ UTR 10 3206 cggcctcggcactggacagc 27 222 111613 3′ UTR10 3277 gtggaatgtctgagatccag 31 223 111614 3′ UTR 10 3322agggcgggcctgcttgccca 23 224 111615 3′ UTR 10 3384 cggtcctggcctgctccaga31 225 111616 3′ UTR 10 3428 tacactgttcccaggagggt 42 226 111617 3′ UTR10 3471 tggtgccagcagcgctagca 10 227 111618 3′ UTR 10 3516cagtctcttcagcctcaaga 43 228 113729 3′ UTR 10 3537 aagagtcatgagcaccatca56 229 111619 3′ UTR 10 3560 tgaaggtcaagttcccctca 40 230 111620 3′ UTR10 3622 ctggcaagaggcagactgga 30 231 111621 3′ UTR 10 3666ggctctgtgctggcttctct 52 232 111622 3′ UTR 10 3711 gccatctcctcagcctgtgc39 233 111623 3′ UTR 10 3787 agcgcctgctctgaggcccc 16 234 111624 3′ UTR10 3854 tgctgagtaagtattgactt 35 235 111625 3′ UTR 10 3927ctatggccatttagagagag 36 236 113730 3′ UTR 10 3936 tggtttattctatggccatt59 237 111626 3′ UTR 10 3994 cgctcctgcaaaggtgctat 11 238 111627 3′ UTR10 4053 gttggaaacggtgcagtcgg 39 239 111628 3′ UTR 10 4095atttattgttgcaactaatg 33 240

[0217] As shown in Table 2, SEQ ID NOs 97, 99, 100, 101, 102, 103, 104,106, 107, 108, 109, 110, 112, 113, 114, 115, 117, 120, 121, 122, 123,124, 126, 127, 128, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 144, 145, 146, 147, 148, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 191, 193, 195, 196, 198, 201, 202, 204, 205, 206, 211, 215, 217,219, 223, 225, 226, 228, 229, 230, 232, 233, 235, 236, 237, 239 and 240demonstrated at least 30% inhibition of rat PTP1B expression in thisexperiment and are therefore preferred.

Example 17 Western Blot Analysis of PTP1B Protein Levels

[0218] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to PTP1B is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18 Effects of Antisense Inhibition of PTP1B (ISIS 113715) onBlood Glucose Levels

[0219] db/db mice are used as a model of Type 2 diabetes. These mice arehyperglycemic, obese, hyperlipidemic, and insulin resistant. The db/dbphenotype is due to a mutation in the leptin receptor on a C57BLKSbackground. However, a mutation in the leptin gene on a different mousebackground can produce obesity without diabetes (ob/ob mice). Leptin isa hormone produced by fat that regulates appetite and animals or humanswith leptin deficiencies become obese. Heterozygous db/wt mice (known aslean littermates) do not display the hyperglycemia/hyperlipidemia orobesity phenotype and are used as controls.

[0220] In accordance with the present invention, ISIS 113715(GCTCCTTCCACTGATCCTGC, SEQ ID No: 166) was investigated in experimentsdesigned to address the role of PTP1B in glucose metabolism andhomeostasis. ISIS 113715 is completely complementary to sequences in thecoding region of the human, rat, and mouse PTP1B nucleotide sequencesincorporated herein as SEQ ID No: 3 (starting at nucleotide 951 of humanPTP1B; Genbank Accession No. M31724), SEQ ID No: 10 (starting atnucleotide 980 of rat PTP1B; Genbank Accession No. M33962) and SEQ IDNo: 241 (starting at nucleotide 1570 of mouse PTP1B; Genbank AccessionNo. U24700). The control used is ISIS 29848 (NNNNNNNNNNNNNNNNNNNN, SEQID No: 242) where N is a mixture of A, G, T and C.

[0221] Male db/db mice and lean (heterozygous, i.e., db/wt) littermates(age 9 weeks at time 0) were divided into matched groups (n=6) with thesame average blood glucose levels and treated by intraperitonealinjection once a week with saline, ISIS 29848 (the controloligonucleotide) or ISIS 113715. db/db mice were treated at a dose of10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg of ISIS 29848 while leanlittermates were treated at a dose of 50 or 100 mg/kg of ISIS 113715 or100 mg/kg of ISIS 29848. Treatment was continued for 4 weeks with bloodglucose levels being measured on day 0, 7, 14, 21 and 28.

[0222] By day 28 in db/db mice, blood glucose levels were reduced at alldoses from a starting level of 300 mg/dL to 225 mg/dL for the 10 mg/kgdose, 175 mg/dL for the 25 mg/kg dose and 125 mg/dL for the 50 mg/kgdose. These final levels are within normal range for wild-type mice (170mg/dL). The mismatch control and saline treated levels levels were 320mg/dL and 370 mg/dL at day 28, respectively.

[0223] In lean littermates, blood glucose levels remained constantthroughout the study for all treatment groups (average 120 mg/dL). Theseresults indicate that treatment with ISIS 113715 reduces blood glucosein db/db mice and that there is no hypoglycemia induced in the db/db orthe lean littermate mice as a result of the oligonucleotide treatment.

[0224] In a similar experiment, ob/ob mice and their lean littermates(heterozygous, i.e., ob/wt) were dosed twice a week at 50 mg/kg withISIS 113715, ISIS 29848 or saline control and blood glucose levels weremeasured at the end of day 7, 14 and 21. Treatment of ob/ob mice withISIS 113715 resulted in the largest decrease in blood glucose over timegoing from 225 mg/dL at day 7 to 95 mg/dL at day 21. Ob/ob micedisplayed an increase in plasma glucose over time from 300 mg/dL to 325mg/dL while treatment with the control oligonucleotide reduced plasmaglucose from an average of 280 mg/dL to 130 mg/dL. In the leanlittermates plasma glucose levels remained unchanged in all treatmentgroups (average level 100 mg/dL).

Example 19 Effects of Antisense Inhibition of PTP1B (ISIS 113715) onmRNA Expression in Liver

[0225] Male db/db mice and lean littermates (age 9 weeks at time 0) weredivided into matched groups (n=6) with the same average blood glucoselevels and treated by intraperitoneal injection once a week with saline,ISIS 29848 (the control oligonucleotide) or ISIS 113715. db/db mice weretreated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg ofISIS 29848 while lean littermates were treated at a dose of 50 or 100mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment was continuedfor 4 weeks after which the mice were sacrificed and tissues collectedfor mRNA analysis. RNA values were normalized and are expressed as apercentage of saline treated control.

[0226] ISIS 113715 successfully reduced PTP1B mRNA levels in the liversof db/db mice at all doses examined (60% reduction of PTP1B mRNA),whereas the control oligonucleotide treated animals showed no reductionin PTP1B mRNA, remaining at the level of the saline treated control.Treatment of lean littermates with ISIS 113715 also reduced mRNA levelsto 45% of control at the 50 mg/kg dose and 25% of control at the 100mg/kg dose. The control oligonucleotide (ISIS 29848) failed to show anyreduction in mRNA levels.

Example 20 Effects of Antisense Inhibition of PTP1B (ISIS 113715) onBody Weight

[0227] Male db/db mice and lean littermates (age 9 weeks at time 0) weredivided into matched groups (n=6) with the same average blood glucoselevels and treated by intraperitoneal injection once a week with saline,ISIS 29848 (the control oligonucleotide) or ISIS 113715. db/db mice weretreated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg ofISIS 29848 while lean littermates were treated at a dose of 50 or 100mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment was continuedfor 4 weeks. At day 28 mice were sacrificed and final body weights weremeasured.

[0228] Treatment of ob/ob mice with ISIS 113715 resulted in an increasein body weight which was constant over the dose range with animalsgaining an average of 11.0 grams while saline treated controls gained5.5 grams. Animals treated with the control oligonucleotide gained anaverage of 7.8 grams of body weight.

[0229] Lean littermate animals treated with 50 or 100 mg/kg of ISIS113715 gained 3.8 grams of body weight compared to a gain of 3.0 gramsfor the saline controls.

[0230] In a similar experiment, ob/ob mice and their lean littermateswere dosed twice a week at 50 mg/kg with ISIS 113715, ISIS 29848 orsaline control and body weights were measured at the end of day 7, 14and 21.

[0231] Treatment of the ob/ob mice with ISIS 113715, ISIS 29848 orsaline control all resulted in a similar increase in body weight acrossthe 21-day timecourse. At the end of day 7 all ob/ob treatment groupshad an average weight of 42 grams. By day 21, animals treated with ISIS113715 had an average body weight of 48 grams, while those in the ISIS29848 (control oligonucleotide) and saline control group each had anaverage body weight of 52 grams. All of the lean littermates had anaverage body weight of 25 grams at the beginning of the timecourse andall lean littermate treatment groups showed an increase in body weight,to 28 grams, by day 21.

Example 21 Effects of Antisense Inhibition of PTP1B (ISIS 113715) onPlasma Insulin Levels

[0232] Male db/db mice (age 9 weeks at time 0) were divided into matchedgroups (n=6) with the same average blood glucose levels and treated byintraperitoneal injection twice a week with saline, ISIS 29848 (thecontrol oligonucleotide) or ISIS 113715 at a dose of 50 mg/kg. Treatmentwas continued for 3 weeks with plasma insulin levels being measured onday 7, 14, and 21.

[0233] Mice treated with ISIS 113715 showed a decrease in plasma insulinlevels from 15 ng/mL at day 7 to 7.5 ng/mL on day 21. Saline treatedanimals has plasma insulin levels of 37 ng/mL at day 7 which dropped to25 ng/mL on day 14 but rose again to 33 ng/mL by day 21. Mice treatedwith the control oligonucleotide also showed a decrease in plasmainsulin levels across the timecourse of the study from 25 ng/mL at day 7to 10 ng/mL on day 21. However, ISIS 113715 was the most effective atreducing plasma insulin over time.

Example 22 Antisense Inhibition of Human PTP1B Expression by AdditionalChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

[0234] In accordance with the present invention, an additional series ofoligonucleotides were designed to target different genomic regions ofthe human PTP1B RNA, using published sequences (GenBank accession numberM31724, incorporated herein as SEQ ID NO: 3), and concatenated genomicsequence derived from nucleotide residues 1-31000 of Genbank accessionnumber AL034429 followed by nucleotide residues 1-45000 of Genbankaccession number AL133230, incorporated herein as SEQ ID NO: 243). Theoligonucleotides are shown in Table 3. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 3 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanPTP1B mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments. If present,“N.D.” indicates “no data”. TABLE 3 Inhibition of human PTP1B mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap Isis # REGION TARGET TARGET SEQUENCE % SEQ ID 1420205′ UTR 3 6 GCGCTCTTAGCCCCGAGGCC 61 244 142021 5′ UTR 3 65CCAGGGCGGCTGCTGCGCCT 56 245 142022 Start 3 80 CATCTCCATGACGGGCCAGG 4 246Codon 142023 Start 3 85 TTTTCCATCTCCATGACGGG 67 247 Codon 142024 Start 390 ACTCCTTTTCCATCTCCATG 71 248 Codon 142025 Exon 1 3 106TTGTCGATCTGCTCGAACTC 61 249 142026 Exon 1 3 109 GACTTGTCGATCTGCTCGAA 66250 142027 Exon 1 3 116 GCTCCCGGACTTGTCGATCT 95 251 142028 Exon 1 3 119CCAGCTCCCGGACTTGTCGA 92 252 142029 Exon:Exon 3 945 TCCACTGATCCTGCACGGAA44 253 Junction 142030 Exon:Exon 3 948 CCTTCCACTGATCCTGCACG 55 254Junction 142031 3′ UTR 3 1453 ATGCCTGCTAGTCGGGCGTG 67 255 142032 3′ UTR3 1670 CGGGTGTAGGCCCCTTCCCT 74 256 142033 3′ UTR 3 1772ATGGAGTGGAGAGTTGCTCC 63 257 142034 3′ UTR 3 1893 TTGTACTTTTTGATAAAGCC 61258 142035 3′ UTR 3 1962 CAGTACTGGTCTGACGCAGC 68 259 142036 3′ UTR 32018 TCTCACGTTACCCACAATAT 74 260 142037 3′ UTR 3 2070TTTCTTATTAAATACCCACG 61 261 142038 3′ UTR 3 2088 AAGTAATCTCACATCATGTT 79262 142039 3′ UTR 3 2314 TTCAGCAACAGGCTTAGGAA 51 263 142040 3′ UTR 32323 GACAATGACTTCAGCAACAG 43 264 142041 3′ UTR 3 2359TGCCTATTCCTGGAAAACTG 43 265 142042 3′ UTR 3 2395 GGAAGTCACTAGAGTGTCAT 14266 142043 3′ UTR 3 2418 CCAGGACAGGCTGGGCCTCA 67 267 142044 3′ UTR 32426 CTGCTGTACCAGGACAGGCT 73 268 142045 3′ UTR 3 2452TGGAATGTCTGAGTTACAGC 74 269 142046 3′ UTR 3 2566 AGAGTGTTGACTTGGAATGG 43270 142047 3′ UTR 3 2574 GCTCAAGAAGAGTGTTGACT 76 271 142048 3′ UTR 32590 TGCCTCTCTTCCAAATCACG 43 272 142049 3′ UTR 3 2800TGTTTTTCATGTTAAAAAGC 44 273 142050 3′ UTR 3 2895 TCCCACCACAGAATTTCTCT 21274 142051 3′ UTR 3 2921 GCTCTGCAGGGTGACACCTC 74 275 142052 3′ UTR 33066 AGGAGGTTAAACCAGTACGT 78 276 142053 3′ UTR 3 3094GGTGGAGAGCCAGCTGCTCT 59 277 142054 3′ UTR 3 3153 TATTGGCTTAAGGCATATAG 72278 142055 3′ UTR 3 3168 GACCTGATGAGTAAATATTG 58 279 142084 5′ UTR 243859 TTCTTCATGTCAACCGGCAG 11 280 142085 5′ UTR 243 919GCCCCGAGGCCCGCTGCAAT 83 281 142056 Intron 1 243 4206TAGTGAACTATTGTTACAAC 70 282 142057 Intron 1 243 27032TGCTAAGCCACTTCTAATCA 72 283 142058 Intron 1 243 27203CAGGATTCTAAGTTATTAAA 32 284 142059 Intron 1 243 33720TGGGCAGGATGGCTCTGGTA 21 285 142060 Intron 1 243 48065TACAATACTATCTGTGACTA 34 286 142061 Exon: 243 51931 GATACTTACAGGGACTGACG39 287 Intron 142086 Intron 2 243 52005 AACCCTGAGGCGAAAGGAGT 64 288142062 Intron 2 243 54384 CCCCAGGTCACTAAAATTAA 48 289 142063 Intron 2243 55362 AAAGCAAAGGTGAGTTGGTG 56 290 142064 Intron 3 243 56093GCTCAATTATTAAACCACTT 64 291 142065 Intron 3 243 56717AGTCCTCAAGAAGTCACTTT 70 292 142066 Intron 4 243 61780GAAAGCAGGGACTGCTGGCA 39 293 142067 Intron 4 243 64554AAAACTGGGAGAGACAGCAG 71 294 142068 Intron 4 243 64869ACATGGAAGCCATGGTCAGC 24 295 142069 Intron 5 243 67516ATTGCTAGACTCACACTAGG 68 296 142070 Intron 5 243 68052GGCTGTGATCAAAAGGCAGC 51 297 142087 Intron 5 243 68481CACTGGCTCTGGGCAACTTT 70 298 142088 Intron 5 243 68563GCTGGGCAGCCACCCATAAA 71 299 142071 Intron 5 243 68648AGTCCCCTCACCTCTTTTCT 59 300 142072 Exon: 243 69107 CCTCCTTACCAGCAAGAGGC26 301 Intron 142089 Intron 6 243 69198 TGTATTTTGGAAGAGGAGCG 53 302142090 Intron 6 243 69220 ACAGACTAACACAGTGAGTC 53 303 142073 Intron 6243 69264 ACAAATTACCGAGTCTCAGG 47 304 142074 Intron 6 243 69472TCATGAAAGGCTTGGTGCCC 41 305 142075 Intron 7 243 70042TTGGAAGATGAAATCTTTTG 30 306 142076 Intron 7 243 70052AGCCATGTACTTGGAAGATG 69 307 142077 Intron 8 243 70661CGAGCCCCTCATTCCAACAA 42 308 142078 Intron 8 243 71005CACCTCAGCGGACACCTCTA 6 309 142079 Exon: 243 71938 GAAACATACCCTGTAGCAGA52 310 Intron 142091 Intron 9 243 72131 CAGAGGGCTCCTTAAAACCC 61 311142092 Intron 9 243 72430 ATTCGTAAAAGTTTGGGATT 34 312 142080 Intron 9243 72453 CCCTCTTCTCCAAGGGAGTT 73 313 142081 Intron 9 243 73158GGAATGAAACCAAACAGTTC 42 314 142082 Exon 10 243 75012AAATGGTTTATTCCATGGCC 66 315 142083 Exon 10 243 75215AAAAATTTTATTGTTGCAGC 48 316 142093 3′ UTR 243 75095 CCGGTCATGCAGCCACGTAT85 317 142094 3′ UTR 243 75165 GTTGGAAAACTGTACAGTCT 77 318 142095 3′ UTR243 75211 ATTTTATTGTTGCAGCTAAA 46 319

[0235] As shown in Table 3, SEQ ID NOs, 244, 245, 247, 248, 249, 250,251, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 267, 268,269, 271, 275, 276, 277, 278, 279, 281, 282, 283, 288, 290, 291, 292,294, 296, 297, 298, 299, 300, 302, 303, 307, 310, 311, 313, 315, 317,and 318, demonstrated at least 50% inhibition of human PTP1B expressionin this assay and are therefore preferred.

Example 23 Antisense Inhibition of Human PTP1B Expression by AdditionalChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

[0236] In accordance with the present invention, an additional series ofoligonucleotides were designed to target either the 3′UTR or the 5′UTRof the human PTP1B RNA, using published sequences (GenBank accessionnumber M31724, incorporated herein as SEQ ID NO: 3) and concatenatedgenomic sequence derived from nucleotide residues 1-31000 of Genbankaccession number AL034429 followed by nucleotide residues 1-45000 ofGenbank accession number AL133230, incorporated herein as SEQ ID NO:243. The oligonucleotides are shown in Table4. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target sequenceto which the oligonucleotide binds. All compounds in Table 3 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanPTP1B mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments. If present,“N.D.” indicates “no data”. TABLE 4 Inhibition of human PTP1B mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap Isis # REGION TARGET TARGET SEQUENCE % SEQ 146879 5′ UTR3 50 CGCCTCCTTCTCGGCCCACT 29 320 146880 5′ UTR 3 62 GGGCGGCTGCTGCGCCTCCT34 321 146881 3′ UTR 3 1601 GTGGATTTGGTACTCAAAGT 72 322 146882 3′ UTR 31610 AAATGGCTTGTGGATTTGGT 72 323 146883 3′ UTR 3 1637ATGGTACTCTCTTTCACTCT 61 324 146884 3′ UTR 3 1643 GCCAGCATGGTACTCTCTTT 63325 146885 3′ UTR 3 1764 GAGAGTTGCTCCCTGCAGAT 62 326 146886 3′ UTR 31770 GGAGTGGAGAGTTGCTCCCT 57 327 146887 3′ UTR 3 1874CCTTGATGCAAGGCTGACAT 65 328 146888 3′ UTR 3 1879 AAAGCCCTTGATGCAAGGCT 59329 146889 3′ UTR 3 1915 AGTACTACCTGAGGATTTAT 46 330 146890 3′ UTR 31925 TTCCATTCCCAGTACTACCT 41 331 146891 3′ UTR 3 1938CCATGGCAAAGCCTTCCATT 65 332 146892 3′ UTR 3 1943 CAGGCCCATGGCAAAGCCTT 52333 146893 3′ UTR 3 1988 CAACTGCTTACAACCGTCCT 60 334 146894 3′ UTR 32055 CCACGTGTTCATTATATATT 42 335 146895 3′ UTR 3 2063TTAAATACCCACGTGTTCAT 27 336 146896 3′ UTR 3 2099 TAAGCGGGACAAAGTAATCT 47337 146897 3′ UTR 3 2118 CAGATAACAGGGAGGAGAAT 31 338 146898 3′ UTR 32133 GAGAACTAGATCTAGCAGAT 0 339 146899 3′ UTR 3 2140AGTGATTGAGAACTAGATCT 62 340 146900 3′ UTR 3 2184 GACACAAGAAGACCTTACAT 49341 146901 3′ UTR 3 2212 CTCATTTCAAGCACATATTT 60 342 146902 3′ UTR 32263 GGCAGGTTGGACTTGGACAT 49 343 146903 3′ UTR 3 2296AACCACAGCCATGTAATGAT 43 344 146904 3′ UTR 3 2332 TTGCTGAGCGACAATGACTT 42345 146905 3′ UTR 3 2350 CTGGAAAACTGCACCCTATT 31 346 146906 3′ UTR 32409 GCTGGGCCTCACCAGGAAGT 77 347 146907 3′ UTR 3 2439TTACAGCAAGACCCTGCTGT 28 348 146908 3′ UTR 3 2457 ACCCTTGGAATGTCTGAGTT 65349 146909 3′ UTR 3 2464 TTCCCATACCCTTGGAATGT 62 350 146910 3′ UTR 32471 ATATGGCTTCCCATACCCTT 47 351 146911 3′ UTR 3 2477GTGTGAATATGGCTTCCCAT 54 352 146912 3′ UTR 3 2509 CCTGCTTCCCTAAATCATGT 65353 146913 3′ UTR 3 2514 GTGTCCCTGCTTCCCTAAAT 55 354 146914 3′ UTR 32546 CGGAGGCTGATCCCAAAGGT 55 355 146915 3′ UTR 3 2602CAGGTGCCTCTCTTCCAAAT 60 356 146916 3′ UTR 3 2613 GTGGTTTCCAGCAGGTGCCT 63357 146917 3′ UTR 3 2628 GCTGTTTCAAGAAGTGTGGT 43 358 146918 3′ UTR 32642 GGACCGTCACCCAGGCTGTT 32 359 146919 3′ UTR 3 2655CAGGCTGCCTAAAGGACCGT 60 360 146920 3′ UTR 3 2732 ACCATCAGGCCCCACAGGGT 58361 146921 3′ UTR 3 2759 GTTCCCTTTGCAGGAAGAGT 69 362 146922 3′ UTR 32772 GTGGAGGTCTTCAGTTCCCT 64 363 146923 3′ UTR 3 2781CCACTTAATGTGGAGGTCTT 54 364 146924 3′ UTR 3 2814 AGCTACAGCTGCCGTGTTTT 51365 146925 3′ UTR 3 2862 CCACGAGAAAGGCAAAATGT 50 366 146926 3′ UTR 32885 GAATTTCTCTGTACTGGCTT 23 367 146927 3′ UTR 3 2890CCACAGAATTTCTCTGTACT 61 368 146928 3′ UTR 3 2901 GAATGTTCCCACCACAGAAT 61369 146929 3′ UTR 3 2956 GCCTGGCACCTAAGCCTTAT 0 370 146930 3′ UTR 3 2965ATGCTTACAGCCTGGCACCT 55 371 146931 3′ UTR 3 3008 CTACATACATATACAGGACT 65372 146932 3′ UTR 3 3042 TTTGAAATGCTACTATATAT 44 373 146933 3′ UTR 33070 GGATAGGAGGTTAAACCAGT 67 374 146934 3′ UTR 3 3086GCCAGCTGCTCTCCAAGGAT 42 375 146935 3′ UTR 3 3121 CTACCTCTCTAACATAATGT 39376 146936 3′ UTR 3 3126 GCTCGCTACCTCTCTAACAT 68 377 146937 3′ UTR 33143 AGGCATATAGCAGAGCAGCT 61 378 146938 5′ UTR 243 851GTCAACCGGCAGCCGGAACT 14 379 146942 5′ UTR 243 891 CCTGCAGCTACCGCCGCCCT69 380 146943 5′ UTR 243 908 CGCTGCAATCCCCGACCCCT 87 381 146944 3′ UTR243 75050 ACCAAAACACCTTGCTTTTT 27 382 146945 3′ UTR 243 75057GTATTATACCAAAACACCTT 39 383 146946 3′ UTR 243 75072 CACACACCTGAAAAGGTATT42 384 146947 3′ UTR 243 75097 ACCCGGTCATGCAGCCACGT 49 385 146948 3′ UTR243 75136 GTGAGGTCACAGAAGACCCT 49 386 146949 3′ UTR 243 75154GTACAGTCTGACAGTTCTGT 40 387 146950 3′ UTR 243 75172 ATGGCAAGTTGGAAAACTGT65 388 146951 3′ UTR 243 75192 AATGCAAACCCATCATGAAT 43 389

[0237] As shown in Table 4, SEQ ID NOs, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 337, 340, 341, 342, 343, 344,345, 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361,362, 363, 364, 365, 366, 368, 369, 371, 372, 373, 374, 375, 377, 378,380, 381, 384, 385, 386, 387, 388, and 389 demonstrated at least 40%inhibition of human PTP1B expression in this assay and are thereforepreferred.

Example 24 Antisense Inhibition of PTP1B Expression (ISIS 113715) inLiver, Muscle and Adipose Tissue of the Cynomolgus Monkey

[0238] In a further embodiment, male cynomolgus monkeys were treatedwith ISIS 113715 (SEQ ID NO: 166) and levels of PTP1B mRNA and proteinwere measured in muscle, adipose and liver tissue. Serum samples werealso measured for insulin levels.

[0239] Male cynomolgus monkeys were divided into two treatment groups,control animals (n=4; saline treatment only) and treated animals (n=8;treated with ISIS 113715). All animals had two pre-dosing glucosetolerance tests (GTTs) performed to establish insulin and glucosebaseline values. Animals in the treatment group were dosedsubcutaneously on days 1, 8, and 15 with 3 mg/kg, 6 mg/kg and 12 mg/kgof ISIS 113715, respectively. Animals in the control group wereuntreated. All animals had GTTs performed on days 5, 13 and 19, fourdays post-dosing. Ten days after the last dose of 12 mg/kg, all animalsin the treatment group (ISIS 113715) received a one-time dose of 6 mg/kgof ISIS 113715. Three days later, all animals were sacrificed andtissues were taken for analysis of PTP1B mRNA and protein levels. Levelsof mRNA and protein were normalized to those of the saline treatedanimals. Of the tissue examined, PTP1B mRNA levels were reduced to thegreatest extent in the fat and liver, being reduced by 41% and 40%,respectively. mRNA levels in muscle were reduced by 10%. Protein levelswere reduced by 60% in the liver and by 45% in the muscle but were shownto increase by 10% in the fat.

[0240] Levels of the liver enzymes ALT and AST were measured weekly andshowed no change, indicating no ongoing toxic effects of theoligonucleotide treatment.

[0241] The results of this study demonstrate a significant reduction inliver PTP1B mRNA and protein upon treatment with ISIS 113715.Furthermore, there was no change seen in the fasting insulin levelseither between groups or between pre-treatment and post-treatment of thesame group. There was, however, a significant lowering of insulin levelswith no decrease in fasting glucose levels in all groups suggesting thatinsulin efficiency (sensitivity) was increased upon treatment with ISIS113715.

Example 25 Effects of Antisense Inhibition of PTP1B (ISIS 113715) onmRNA Expression in Fractionated Liver

[0242] Male db/db mice (age 9 weeks at time 0) were divided into matchedgroups (n=6) with the same average blood glucose levels and treated byintraperitoneal injection once a week with saline, ISIS 29848 (thecontrol oligonucleotide) or ISIS 113715. db/db mice were treated at adose of 50 mg/kg of ISIS 113715 or 50 mg/kg of ISIS 29848 or 100 mg/kgof ISIS 29848. Treatment was continued for 3 weeks after which the micewere sacrificed and tissues were collected for analysis. Liver tissuewas removed and homogenized whole or fractionated into hepatocytes andnon-parenchymal (NP) cell fractions by standard methods (Graham et al.,J. Pharmacol. Exp. Ther., 1998, 286, 447-458). During the study, plasmaglucose levels were measured as were PTP1B mRNA levels in both cellfractions. RNA values were normalized and are expressed as a percentageof saline treated control.

[0243] Treatment of db/db mice with ISIS 113715 caused a significantreduction in plasma glucose levels (saline=500+/−25 vs. treated=223+/−21mg/dL; p=0.0001).

[0244] ISIS 113715 successfully reduced PTP1B mRNA levels in bothhepatocytes and NP cell fractions, with an 80% reduction in hepatocytesand a 30% reduction in the NP cell fraction. In addition, PTP1Bexpression in the two cell fractions was found to be dramaticallydifferent with a 5-8 fold greater level of expression being found in theNP fraction. Thus, the inability of ISIS 113715 to reduce PTP1Bexpression by no more than 60% in whole liver as seen in previousexperiments may result from a combination of a relatively highexpression of PTP1B in NP cells with a reduced ability of ISIS 113715 toinhibit expression in this same cell fraction. Consequently, distincttargeting of the compound to hepatocytes, the key metabolic cell type inliver, results in a much greater inhibition of PTP1B levels.

Example 26 Effects of Antisense Inhibition of PTP1B Expression (ISIS113715) in the Obese Insulin-resistant Hyperinsulinemic Rhesusmonkey-Improved Insulin Sensitivity

[0245] In a further embodiment, male obese insulin-resistanthyperinsulinemic Rhesus monkeys were treated with ISIS 113715 (SEQ IDNO: 166) and insulin sensitivity, glucose tolerance and PTP1B mRNA andprotein were measured. Serum samples were also measured for insulinlevels.

[0246] Male rhesus monkeys were divided into two treatment groups,control animals (n=4; saline treatment only) and treated animals (n=8;treated with ISIS 113715). All animals had two pre-dosing glucosetolerance tests (GTTs) performed to establish insulin and glucosebaseline values. Animals in the treatment group were dosedsubcutaneously at a dose of 20 mg/kg (3 injections on alternate days thefirst week followed by one injection per week for the next two weeks).Fasted glucose/insulin levels and glucose tolerance (IVGTTs) weremeasured as pharmacologic endpoints.

[0247] As compared to baseline values, a 50% reduction in fastinginsulin levels was observed (treated: 31±9 vs. baseline: 67±7 uU/mL,p=0.0001), which was not accompanied by any change in plasma glucoselevels. Furthermore, a marked reduction in insulin levels (AUC) wasobserved after IVGTTs (treated: 7295±3178 vs. baseline: 18968±2113μU-min/mL, p=0.0002). Insulin sensitivity was also significantlyincreased (glucose slope/insulin AUC; 5-20 minutes).

[0248] Hypoglycemia was not observed, even in the 16 hour-fastedanimals. Levels of the liver enzymes ALT and AST were measured weeklyand showed no change, indicating no ongoing toxic effects of theoligonucleotide treatment. Renal function tests were also normal.

[0249] The results of this study are consistent with those seen inprevious rodent and monkey studies described herein which demonstrate asignificant lowering of insulin levels suggesting that insulinefficiency (sensitivity) was increased upon treatment with ISIS 113715.

Example 27 RNA Synthesis

[0250] In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

[0251] Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

[0252] RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

[0253] Following synthesis, the methyl protecting groups on thephosphates are cleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1, 1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

[0254] The 2′-orthoester groups are the last protecting groups to beremoved. The ethylene glycol monoacetate orthoester protecting groupdeveloped by Dharmacon Research, Inc. (Lafayette, Colo.), is one exampleof a useful orthoester protecting group which, has the followingimportant properties. It is stable to the conditions of nucleosidephosphoramidite synthesis and oligonucleotide synthesis. However, afteroligonucleotide synthesis the oligonucleotide is treated withmethylamine which not only cleaves the oligonucleotide from the solidsupport but also removes the acetyl groups from the orthoesters. Theresulting 2-ethyl-hydroxyl substituents on the orthoester are lesselectron withdrawing than the acetylated precursor. As a result, themodified orthoester becomes more labile to acid-catalyzed hydrolysis.Specifically, the rate of cleavage is approximately 10 times fasterafter the acetyl groups are removed. Therefore, this orthoesterpossesses sufficient stability in order to be compatible witholigonucleotide synthesis and yet, when subsequently modified, permitsdeprotection to be carried out under relatively mild aqueous conditionscompatible with the final RNA oligonucleotide product.

[0255] Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

[0256] RNA antisense compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense compounds can then be annealed by methodsknown in the art to form double stranded (duplexed) antisense compounds.For example, duplexes can be formed by combining 30 μl of each of thecomplementary strands of RNA oligonucleotides (50 uM RNA oligonucleotidesolution) and 15 μl of 5×annealing buffer (100 mM potassium acetate, 30mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisensecompounds can be used in kits, assays, screens, or other methods toinvestigate the role of a target nucleic acid.

Example 28 Design and Screening of Duplexed Antisense CompoundsTargeting PTP1B

[0257] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target PTP1B. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

[0258] For example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

[0259] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 uM. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5×solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0260] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate PTP1B expression. When cells reached 80%confluency, they are treated with duplexed antisense compounds of theinvention. For cells grown in 96-well plates, wells are washed once with200 uL OPTI-MEMT™-1 reduced-serum medium (Gibco BRL) and then treatedwith 130 uL of OPTI-MEM™-1 containing 12 ug/mL LIPOFECTIN™ (Gibco BRL)and the desired duplex antisense compound at a final concentration of200 nM. After 5 hours of treatment, the medium is replaced with freshmedium. Cells are harvested 16 hours after treatment, at which time RNAis isolated and target reduction measured by RT-PCR.

1 389 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 atgcattctg cccccaagga 20 3 3247 DNA Homo sapiens CDS(91)...(1398) 3 gggcgggcct cggggctaag agcgcgacgc ctagagcggc agacggcgcagtgggccgag 60 aaggaggcgc agcagccgcc ctggcccgtc atg gag atg gaa aag gagttc gag 114 Met Glu Met Glu Lys Glu Phe Glu 1 5 cag atc gac aag tcc gggagc tgg gcg gcc att tac cag gat atc cga 162 Gln Ile Asp Lys Ser Gly SerTrp Ala Ala Ile Tyr Gln Asp Ile Arg 10 15 20 cat gaa gcc agt gac ttc ccatgt aga gtg gcc aag ctt cct aag aac 210 His Glu Ala Ser Asp Phe Pro CysArg Val Ala Lys Leu Pro Lys Asn 25 30 35 40 aaa aac cga aat agg tac agagac gtc agt ccc ttt gac cat agt cgg 258 Lys Asn Arg Asn Arg Tyr Arg AspVal Ser Pro Phe Asp His Ser Arg 45 50 55 att aaa cta cat caa gaa gat aatgac tat atc aac gct agt ttg ata 306 Ile Lys Leu His Gln Glu Asp Asn AspTyr Ile Asn Ala Ser Leu Ile 60 65 70 aaa atg gaa gaa gcc caa agg agt tacatt ctt acc cag ggc cct ttg 354 Lys Met Glu Glu Ala Gln Arg Ser Tyr IleLeu Thr Gln Gly Pro Leu 75 80 85 cct aac aca tgc ggt cac ttt tgg gag atggtg tgg gag cag aaa agc 402 Pro Asn Thr Cys Gly His Phe Trp Glu Met ValTrp Glu Gln Lys Ser 90 95 100 agg ggt gtc gtc atg ctc aac aga gtg atggag aaa ggt tcg tta aaa 450 Arg Gly Val Val Met Leu Asn Arg Val Met GluLys Gly Ser Leu Lys 105 110 115 120 tgc gca caa tac tgg cca caa aaa gaagaa aaa gag atg atc ttt gaa 498 Cys Ala Gln Tyr Trp Pro Gln Lys Glu GluLys Glu Met Ile Phe Glu 125 130 135 gac aca aat ttg aaa tta aca ttg atctct gaa gat atc aag tca tat 546 Asp Thr Asn Leu Lys Leu Thr Leu Ile SerGlu Asp Ile Lys Ser Tyr 140 145 150 tat aca gtg cga cag cta gaa ttg gaaaac ctt aca acc caa gaa act 594 Tyr Thr Val Arg Gln Leu Glu Leu Glu AsnLeu Thr Thr Gln Glu Thr 155 160 165 cga gag atc tta cat ttc cac tat accaca tgg cct gac ttt gga gtc 642 Arg Glu Ile Leu His Phe His Tyr Thr ThrTrp Pro Asp Phe Gly Val 170 175 180 cct gaa tca cca gcc tca ttc ttg aacttt ctt ttc aaa gtc cga gag 690 Pro Glu Ser Pro Ala Ser Phe Leu Asn PheLeu Phe Lys Val Arg Glu 185 190 195 200 tca ggg tca ctc agc ccg gag cacggg ccc gtt gtg gtg cac tgc agt 738 Ser Gly Ser Leu Ser Pro Glu His GlyPro Val Val Val His Cys Ser 205 210 215 gca ggc atc ggc agg tct gga accttc tgt ctg gct gat acc tgc ctc 786 Ala Gly Ile Gly Arg Ser Gly Thr PheCys Leu Ala Asp Thr Cys Leu 220 225 230 ctg ctg atg gac aag agg aaa gaccct tct tcc gtt gat atc aag aaa 834 Leu Leu Met Asp Lys Arg Lys Asp ProSer Ser Val Asp Ile Lys Lys 235 240 245 gtg ctg tta gaa atg agg aag tttcgg atg ggg ttg atc cag aca gcc 882 Val Leu Leu Glu Met Arg Lys Phe ArgMet Gly Leu Ile Gln Thr Ala 250 255 260 gac cag ctg cgc ttc tcc tac ctggct gtg atc gaa ggt gcc aaa ttc 930 Asp Gln Leu Arg Phe Ser Tyr Leu AlaVal Ile Glu Gly Ala Lys Phe 265 270 275 280 atc atg ggg gac tct tcc gtgcag gat cag tgg aag gag ctt tcc cac 978 Ile Met Gly Asp Ser Ser Val GlnAsp Gln Trp Lys Glu Leu Ser His 285 290 295 gag gac ctg gag ccc cca cccgag cat atc ccc cca cct ccc cgg cca 1026 Glu Asp Leu Glu Pro Pro Pro GluHis Ile Pro Pro Pro Pro Arg Pro 300 305 310 ccc aaa cga atc ctg gag ccacac aat ggg aaa tgc agg gag ttc ttc 1074 Pro Lys Arg Ile Leu Glu Pro HisAsn Gly Lys Cys Arg Glu Phe Phe 315 320 325 cca aat cac cag tgg gtg aaggaa gag acc cag gag gat aaa gac tgc 1122 Pro Asn His Gln Trp Val Lys GluGlu Thr Gln Glu Asp Lys Asp Cys 330 335 340 ccc atc aag gaa gaa aaa ggaagc ccc tta aat gcc gca ccc tac ggc 1170 Pro Ile Lys Glu Glu Lys Gly SerPro Leu Asn Ala Ala Pro Tyr Gly 345 350 355 360 atc gaa agc atg agt caagac act gaa gtt aga agt cgg gtc gtg ggg 1218 Ile Glu Ser Met Ser Gln AspThr Glu Val Arg Ser Arg Val Val Gly 365 370 375 gga agt ctt cga ggt gcccag gct gcc tcc cca gcc aaa ggg gag ccg 1266 Gly Ser Leu Arg Gly Ala GlnAla Ala Ser Pro Ala Lys Gly Glu Pro 380 385 390 tca ctg ccc gag aag gacgag gac cat gca ctg agt tac tgg aag ccc 1314 Ser Leu Pro Glu Lys Asp GluAsp His Ala Leu Ser Tyr Trp Lys Pro 395 400 405 ttc ctg gtc aac atg tgcgtg gct acg gtc ctc acg gcc ggc gct tac 1362 Phe Leu Val Asn Met Cys ValAla Thr Val Leu Thr Ala Gly Ala Tyr 410 415 420 ctc tgc tac agg ttc ctgttc aac agc aac aca tag cctgaccctc 1408 Leu Cys Tyr Arg Phe Leu Phe AsnSer Asn Thr 425 430 435 ctccactcca cctccaccca ctgtccgcct ctgcccgcagagcccacgcc cgactagcag 1468 gcatgccgcg gtaggtaagg gccgccggac cgcgtagagagccgggcccc ggacggacgt 1528 tggttctgca ctaaaaccca tcttccccgg atgtgtgtctcacccctcat ccttttactt 1588 tttgcccctt ccactttgag taccaaatcc acaagccattttttgaggag agtgaaagag 1648 agtaccatgc tggcggcgca gagggaaggg gcctacacccgtcttggggc tcgccccacc 1708 cagggctccc tcctggagca tcccaggcgg cgcacgccaacagccccccc cttgaatctg 1768 cagggagcaa ctctccactc catatttatt taaacaattttttccccaaa ggcatccata 1828 gtgcactagc attttcttga accaataatg tattaaaattttttgatgtc agccttgcat 1888 caagggcttt atcaaaaagt acaataataa atcctcaggtagtactggga atggaaggct 1948 ttgccatggg cctgctgcgt cagaccagta ctgggaaggaggacggttgt aagcagttgt 2008 tatttagtga tattgtgggt aacgtgagaa gatagaacaatgctataata tataatgaac 2068 acgtgggtat ttaataagaa acatgatgtg agattactttgtcccgctta ttctcctccc 2128 tgttatctgc tagatctagt tctcaatcac tgctcccccgtgtgtattag aatgcatgta 2188 aggtcttctt gtgtcctgat gaaaaatatg tgcttgaaatgagaaacttt gatctctgct 2248 tactaatgtg ccccatgtcc aagtccaacc tgcctgtgcatgacctgatc attacatggc 2308 tgtggttcct aagcctgttg ctgaagtcat tgtcgctcagcaatagggtg cagttttcca 2368 ggaataggca tttgctaatt cctggcatga cactctagtgacttcctggt gaggcccagc 2428 ctgtcctggt acagcagggt cttgctgtaa ctcagacattccaagggtat gggaagccat 2488 attcacacct cacgctctgg acatgattta gggaagcagggacacccccc gccccccacc 2548 tttgggatca gcctccgcca ttccaagtca acactcttcttgagcagacc gtgatttgga 2608 agagaggcac ctgctggaaa ccacacttct tgaaacagcctgggtgacgg tcctttaggc 2668 agcctgccgc cgtctctgtc ccggttcacc ttgccgagagaggcgcgtct gccccaccct 2728 caaaccctgt ggggcctgat ggtgctcacg actcttcctgcaaagggaac tgaagacctc 2788 cacattaagt ggctttttaa catgaaaaac acggcagctgtagctcccga gctactctct 2848 tgccagcatt ttcacatttt gcctttctcg tggtagaagccagtacagag aaattctgtg 2908 gtgggaacat tcgaggtgtc accctgcaga gctatggtgaggtgtggata aggcttaggt 2968 gccaggctgt aagcattctg agctggcttg ttgtttttaagtcctgtata tgtatgtagt 3028 agtttgggtg tgtatatata gtagcatttc aaaatggacgtactggttta acctcctatc 3088 cttggagagc agctggctct ccaccttgtt acacattatgttagagaggt agcgagctgc 3148 tctgctatat gccttaagcc aatatttact catcaggtcattatttttta caatggccat 3208 ggaataaacc atttttacaa aaataaaaac aaaaaaagc3247 4 21 DNA Artificial Sequence PCR Primer 4 ggagttcgag cagatcgaca a21 5 21 DNA Artificial Sequence PCR Primer 5 ggccactcta catgggaagt c 216 24 DNA Artificial Sequence PCR Probe 6 agctgggcgg ccatttacca ggat 24 719 DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNAArtificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNAArtificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 4127 DNARattus norvegicus CDS (120)...(1418) 10 agccgctgct ggggaggttg gggctgaggtggtggcgggc gacgggcctc gagacgcgga 60 gcgacgcggc ctagcgcggc ggacggccgagggaactcgg gcagtcgtcc cgtcccgcc 119 atg gaa atg gag aag gaa ttc gag cagatc gat aag gct ggg aac tgg 167 Met Glu Met Glu Lys Glu Phe Glu Gln IleAsp Lys Ala Gly Asn Trp 1 5 10 15 gcg gct att tac cag gat att cga catgaa gcc agt gac ttc cca tgc 215 Ala Ala Ile Tyr Gln Asp Ile Arg His GluAla Ser Asp Phe Pro Cys 20 25 30 aga ata gcg aaa ctt cct aag aac aaa aaccgg aac agg tac cga gat 263 Arg Ile Ala Lys Leu Pro Lys Asn Lys Asn ArgAsn Arg Tyr Arg Asp 35 40 45 gtc agc cct ttt gac cac agt cgg att aaa ttgcat cag gaa gat aat 311 Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu HisGln Glu Asp Asn 50 55 60 gac tat atc aat gcc agc ttg ata aaa atg gag gaagcc cag agg agc 359 Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu AlaGln Arg Ser 65 70 75 80 tat atc ctc acc cag ggc cct tta cca aac acg tgcggg cac ttc tgg 407 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys GlyHis Phe Trp 85 90 95 gag atg gtg tgg gag cag aag agc agg ggc gtg gtc atgctc aac cgc 455 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met LeuAsn Arg 100 105 110 atc atg gag aaa ggc tcg tta aaa tgt gcc cag tat tggcca cag aaa 503 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp ProGln Lys 115 120 125 gaa gaa aaa gag atg gtc ttc gat gac acc aat ttg aagctg aca ctg 551 Glu Glu Lys Glu Met Val Phe Asp Asp Thr Asn Leu Lys LeuThr Leu 130 135 140 atc tct gaa gat gtc aag tca tat tac aca gta cgg cagttg gag ttg 599 Ile Ser Glu Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln LeuGlu Leu 145 150 155 160 gag aac ctg gct acc cag gag gct cga gag atc ctgcat ttc cac tac 647 Glu Asn Leu Ala Thr Gln Glu Ala Arg Glu Ile Leu HisPhe His Tyr 165 170 175 acc acc tgg cct gac ttt gga gtc cct gag tca cctgcc tct ttc ctc 695 Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro AlaSer Phe Leu 180 185 190 aat ttc cta ttc aaa gtc cga gag tca ggc tca ctcagc cca gag cac 743 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu SerPro Glu His 195 200 205 ggc ccc att gtg gtc cac tgc agt gct ggc att ggcagg tca ggg acc 791 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly ArgSer Gly Thr 210 215 220 ttc tgc ctg gct gac acc tgc ctc tta ctg atg gacaag agg aaa gac 839 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp LysArg Lys Asp 225 230 235 240 ccg tcc tct gtg gac atc aag aaa gtg ctg ttggag atg cgc agg ttc 887 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu GluMet Arg Arg Phe 245 250 255 cgc atg ggg ctc atc cag acg gcc gac caa ctgcgc ttc tcc tac ctg 935 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu ArgPhe Ser Tyr Leu 260 265 270 gct gtg atc gag ggt gca aag ttc atc atg ggcgac tcg tca gtg cag 983 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly AspSer Ser Val Gln 275 280 285 gat cag tgg aag gag ctt tcc cat gaa gac ctggag cct ccc cct gag 1031 Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu GluPro Pro Pro Glu 290 295 300 cac gtg ccc cca cct ccc cgg cca ccc aaa cgcaca ttg gag cct cac 1079 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg ThrLeu Glu Pro His 305 310 315 320 aat ggc aag tgc aag gag ctc ttc tcc aaccac cag tgg gtg agc gag 1127 Asn Gly Lys Cys Lys Glu Leu Phe Ser Asn HisGln Trp Val Ser Glu 325 330 335 gag agc tgt gag gat gag gac atc ctg gccaga gag gaa agc aga gcc 1175 Glu Ser Cys Glu Asp Glu Asp Ile Leu Ala ArgGlu Glu Ser Arg Ala 340 345 350 ccc tca att gct gtg cac agc atg agc agtatg agt caa gac act gaa 1223 Pro Ser Ile Ala Val His Ser Met Ser Ser MetSer Gln Asp Thr Glu 355 360 365 gtt agg aaa cgg atg gtg ggt gga ggt cttcaa agt gct cag gca tct 1271 Val Arg Lys Arg Met Val Gly Gly Gly Leu GlnSer Ala Gln Ala Ser 370 375 380 gtc ccc act gag gaa gag ctg tcc cca accgag gag gaa caa aag gca 1319 Val Pro Thr Glu Glu Glu Leu Ser Pro Thr GluGlu Glu Gln Lys Ala 385 390 395 400 cac agg cca gtt cac tgg aag ccc ttcctg gtc aac gtg tgc atg gcc 1367 His Arg Pro Val His Trp Lys Pro Phe LeuVal Asn Val Cys Met Ala 405 410 415 acg gcc ctg gcg act ggc gcg tac ctctgt tac cgg gta tgt ttt cac 1415 Thr Ala Leu Ala Thr Gly Ala Tyr Leu CysTyr Arg Val Cys Phe His 420 425 430 tga cagactgctg tgaggcatga gcgtggtgggcgctgccact gcccaggtta 1468 ggatttggtc tgcggcgtct aacctggtgt agaagaaacaacagcttaca agcctgtggt 1528 ggaactggaa gggccagccc caggaggggc atctgtgcactgggctttga aggagcccct 1588 ggtcccaaga acagagtcta atctcagggc cttaacctgttcaggagaag tagaggaaat 1648 gccaaatact cttcttgctc tcacctcact cctcccctttctctggttcg tttgtttttg 1708 gaaaaaaaaa aaaaagaatt acaacacatt gttgtttttaacatttataa aggcaggttt 1768 ttgttatttt tagagaaaac aaaagatgct aggcactggtgagattctct tgtgcccttt 1828 ggcatgtgat cagattcacg atttacgttt atttccgggggagggtccca cctgtcagga 1888 ctgtaaagtt cctgctggct tggtcagccc ccccacccccccaccccgag cttgcaggtg 1948 ccctgctgtg aggagagcag cagcagaggc tgcccctggacagaagccca gctctgcttc 2008 cctcaggtgt ccctgcgttt ccatcctcct tctttgtgaccgccatcttg cagatgaccc 2068 agtcctcagc accccacccc tgcagatggg tttctccgagggcctgcctc agggtcatca 2128 gaggttggct gccagcttag agctggggct tccatttgattggaaagtca ttactattct 2188 atgtagaagc cactccactg aggtgtaaag caagactcataaaggaggag ccttggtgtc 2248 atggaagtca ctccgcgcgc aggacctgta acaacctctgaaacactcag tcctgctgca 2308 gtgacgtcct tgaaggcatc agacagatga tttgcagactgccaagactt gtcctgagcc 2368 gtgattttta gagtctggac tcatgaaaca ccgccgagcgcttactgtgc agcctctgat 2428 gctggttggc tgaggctgcg gggaggtgga cactgtgggtgcatccagtg cagttgcttt 2488 tgtgcagttg ggtccagcag cacagcccgc actccagcctcagctgcagg ccacagtggc 2548 catggaggcc gccagagcga gctggggtgg atgcttgttcacttggagca gccttcccag 2608 gacgtgcagc tcccttcctg ctttgtcctt ctgcttccttccctggagta gcaagcccac 2668 gagcaatcgt gaggggtgtg agggagctgc agaggcatcagagtggcctg cagcggcgtg 2728 aggccccttc ccctccgaca cccccctcca gaggagccgctccactgtta tttattcact 2788 ttgcccacag acacccctga gtgagcacac cctgaaactgaccgtgtaag gtgtcagcct 2848 gcacccagga ccgtcaggtg cagcaccggg tcagtcctagggttgaggta ggactgacac 2908 agccactgtg tggctggtgc tggggcaggg gcaggagctgagggtcttag aagcaatctt 2968 caggaacaga caacagtggt gacatgtaaa gtccctgtggctactgatga catgtgtagg 3028 atgaaggctg gcctttctcc catgactttc tagatcccgttccccgtctg ctttccctgt 3088 gagttagaaa acacacaggc tcctgtcctg gtggtgccgtgtgcttgaca tgggaaactt 3148 agatgcctgc tcactggcgg gcacctcggc atcgccaccactcagagtga gagcagtgct 3208 gtccagtgcc gaggccgcct gactcccggc aggactcttcaggctctggc ctgccccagc 3268 acaccccgct ggatctcaga cattccacac ccacacctcattccctggac acttgggcaa 3328 gcaggcccgc ccttccacct ctggggtcag cccctccattccgagttcac actgctctgg 3388 agcaggccag gaccggaagc aaggcagctg gtgaggagcaccctcctggg aacagtgtag 3448 gtgacagtcc tgagagtcag cttgctagcg ctgctggcaccagtcacctt gctcagaagt 3508 gtgtggctct tgaggctgaa gagactgatg atggtgctcatgactcttct gtgaggggaa 3568 cttgaccttc acattgggtg gcttttttta aaataagcgaaggcagctgg aactccagtc 3628 tgcctcttgc cagcacttca cattttgcct ttcacccagagaagccagca cagagccact 3688 ggggaaggcg atggccttgc ctgcacaggc tgaggagatggctcagccgg cgtccaggct 3748 gtgtctggag cagggggtgc acagcagcct cacaggtgggggcctcagag caggcgctgc 3808 cctgtcccct gccccgctgg aggcagcaaa gctgctgcatgccttaagtc aatacttact 3868 cagcagggcg ctctcgttct ctctctctct ctctctctctctctctctct ctctctctct 3928 ctctctaaat ggccatagaa taaaccattt tacaaaaataaaagccaaca acaaagtgct 3988 ctggaatagc acctttgcag gagcgggggg tgtctcagggtcttctgtga cctcaccgaa 4048 ctgtccgact gcaccgtttc caacttgtgt ctcactaatgggtctgcatt agttgcaaca 4108 ataaatgttt ttaaagaac 4127 11 21 DNAArtificial Sequence PCR Primer 11 cgagggtgca aagttcatca t 21 12 21 DNAArtificial Sequence PCR Primer 12 ccaggtcttc atgggaaagc t 21 13 26 DNAArtificial Sequence PCR Probe 13 cgactcgtca gtgcaggatc agtgga 26 14 23DNA Artificial Sequence PCR Primer 14 tgttctagag acagccgcat ctt 23 15 21DNA Artificial Sequence PCR Primer 15 caccgacctt caccatcttg t 21 16 24DNA Artificial Sequence PCR Probe 16 ttgtgcagtg ccagcctcgt ctca 24 17 20DNA Artificial Sequence Antisense Oligonucleotide 17 cttagccccgaggcccgccc 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18ctcggcccac tgcgccgtct 20 19 20 DNA Artificial Sequence AntisenseOligonucleotide 19 catgacgggc cagggcggct 20 20 20 DNA ArtificialSequence Antisense Oligonucleotide 20 cccggacttg tcgatctgct 20 21 20 DNAArtificial Sequence Antisense Oligonucleotide 21 ctggcttcat gtcggatatc20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 ttggccactctacatgggaa 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23ggactgacgt ctctgtacct 20 24 20 DNA Artificial Sequence AntisenseOligonucleotide 24 gatgtagttt aatccgacta 20 25 20 DNA ArtificialSequence Antisense Oligonucleotide 25 ctagcgttga tatagtcatt 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 gggtaagaat gtaactcctt20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tgaccgcatgtgttaggcaa 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28ttttctgctc ccacaccatc 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 ctctgttgag catgacgaca 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 gcgcatttta acgaaccttt 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 aaatttgtgt cttcaaagat20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tgatatcttcagagatcaat 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33tctagctgtc gcactgtata 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 agtttcttgg gttgtaaggt 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 gtggtatagt ggaaatgtaa 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 tgattcaggg actccaaagt20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 ttgaaaagaaagttcaagaa 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38gggctgagtg accctgactc 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 gcagtgcacc acaacgggcc 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 aggttccaga cctgccgatg 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 agcaggaggc aggtatcagc20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 gaagaagggtctttcctctt 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43tctaacagca ctttcttgat 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 atcaacccca tccgaaactt 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 gagaagcgca gctggtcggc 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 tttggcacct tcgatcacag20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 agctccttccactgatcctg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48tccaggattc gtttgggtgg 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 gaactccctg catttcccat 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 ttccttcacc cactggtgat 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 gtagggtgcg gcatttaagg20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 cagtgtcttgactcatgctt 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53gcctgggcac ctcgaagact 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 ctcgtccttc tcgggcagtg 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 gggcttccag taactcagtg 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 ccgtagccac gcacatgttg20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 tagcagaggtaagcgccggc 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58ctatgtgttg ctgttgaaca 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 ggaggtggag tggaggaggg 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 ggctctgcgg gcagaggcgg 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 ccgcggcatg cctgctagtc20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 tctctacgcggtccggcggc 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63aagatgggtt ttagtgcaga 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 gtactctctt tcactctcct 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 ggccccttcc ctctgcgccg 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 ctccaggagg gagccctggg20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 gggctgttggcgtgcgccgc 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68tttaaataaa tatggagtgg 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 gttcaagaaa atgctagtgc 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 ttgataaagc ccttgatgca 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 atggcaaagc cttccattcc20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 gtcctccttcccagtactgg 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73ttacccacaa tatcactaaa 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 attatatatt atagcattgt 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 tcacatcatg tttcttatta 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 ataacaggga ggagaataag20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ttacatgcattctaatacac 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78gatcaaagtt tctcatttca 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 ggtcatgcac aggcaggttg 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 caacaggctt aggaaccaca 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 aactgcaccc tattgctgag20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 gtcatgccaggaattagcaa 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83acaggctggg cctcaccagg 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 tgagttacag caagaccctg 20 85 20 DNA ArtificialSequence Antisense Oligonucleotide 85 gaatatggct tcccataccc 20 86 20 DNAArtificial Sequence Antisense Oligonucleotide 86 ccctaaatca tgtccagagc20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 gacttggaatggcggaggct 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88caaatcacgg tctgctcaag 20 89 20 DNA Artificial Sequence AntisenseOligonucleotide 89 gaagtgtggt ttccagcagg 20 90 20 DNA ArtificialSequence Antisense Oligonucleotide 90 cctaaaggac cgtcacccag 20 91 20 DNAArtificial Sequence Antisense Oligonucleotide 91 gtgaaccggg acagagacgg20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 gccccacagggtttgagggt 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93cctttgcagg aagagtcgtg 20 94 20 DNA Artificial Sequence AntisenseOligonucleotide 94 aaagccactt aatgtggagg 20 95 20 DNA ArtificialSequence Antisense Oligonucleotide 95 gtgaaaatgc tggcaagaga 20 96 20 DNAArtificial Sequence Antisense Oligonucleotide 96 tcagaatgct tacagcctgg20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 caacctccccagcagcggct 20 98 20 DNA Artificial Sequence Antisense Oligonucleotide 98tcgaggcccg tcgcccgcca 20 99 20 DNA Artificial Sequence AntisenseOligonucleotide 99 cctcggccgt ccgccgcgct 20 100 20 DNA ArtificialSequence Antisense Oligonucleotide 100 tcgatctgct cgaattcctt 20 101 20DNA Artificial Sequence Antisense Oligonucleotide 101 cctggtaaatagccgcccag 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide102 tgtcgaatat cctggtaaat 20 103 20 DNA Artificial Sequence AntisenseOligonucleotide 103 actggcttca tgtcgaatat 20 104 20 DNA ArtificialSequence Antisense Oligonucleotide 104 aagtcactgg cttcatgtcg 20 105 20DNA Artificial Sequence Antisense Oligonucleotide 105 gaagtcactggcttcatgtc 20 106 20 DNA Artificial Sequence Antisense Oligonucleotide106 ggaagtcact ggcttcatgt 20 107 20 DNA Artificial Sequence AntisenseOligonucleotide 107 gggaagtcac tggcttcatg 20 108 20 DNA ArtificialSequence Antisense Oligonucleotide 108 tgggaagtca ctggcttcat 20 109 20DNA Artificial Sequence Antisense Oligonucleotide 109 atgggaagtcactggcttca 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide110 catgggaagt cactggcttc 20 111 20 DNA Artificial Sequence AntisenseOligonucleotide 111 tttttgttct taggaagttt 20 112 20 DNA ArtificialSequence Antisense Oligonucleotide 112 cggtttttgt tcttaggaag 20 113 20DNA Artificial Sequence Antisense Oligonucleotide 113 tccgactgtggtcaaaaggg 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide114 ttaatccgac tgtggtcaaa 20 115 20 DNA Artificial Sequence AntisenseOligonucleotide 115 atagtcatta tcttcctgat 20 116 20 DNA ArtificialSequence Antisense Oligonucleotide 116 ttgatatagt cattatcttc 20 117 20DNA Artificial Sequence Antisense Oligonucleotide 117 gcttcctccatttttatcaa 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide118 ggccctgggt gaggatatag 20 119 20 DNA Artificial Sequence AntisenseOligonucleotide 119 cacaccatct cccagaagtg 20 120 20 DNA ArtificialSequence Antisense Oligonucleotide 120 tgctcccaca ccatctccca 20 121 20DNA Artificial Sequence Antisense Oligonucleotide 121 ctgctcccacaccatctccc 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide122 tctgctccca caccatctcc 20 123 20 DNA Artificial Sequence AntisenseOligonucleotide 123 ttctgctccc acaccatctc 20 124 20 DNA ArtificialSequence Antisense Oligonucleotide 124 cccctgctct tctgctccca 20 125 20DNA Artificial Sequence Antisense Oligonucleotide 125 atgcggttgagcatgaccac 20 126 20 DNA Artificial Sequence Antisense Oligonucleotide126 tttaacgagc ctttctccat 20 127 20 DNA Artificial Sequence AntisenseOligonucleotide 127 ttttcttctt tctgtggcca 20 128 20 DNA ArtificialSequence Antisense Oligonucleotide 128 gaccatctct ttttcttctt 20 129 20DNA Artificial Sequence Antisense Oligonucleotide 129 tcagagatcagtgtcagctt 20 130 20 DNA Artificial Sequence Antisense Oligonucleotide130 cttgacatct tcagagatca 20 131 20 DNA Artificial Sequence AntisenseOligonucleotide 131 taatatgact tgacatcttc 20 132 20 DNA ArtificialSequence Antisense Oligonucleotide 132 aactccaact gccgtactgt 20 133 20DNA Artificial Sequence Antisense Oligonucleotide 133 tctctcgagcctcctgggta 20 134 20 DNA Artificial Sequence Antisense Oligonucleotide134 ccaaagtcag gccaggtggt 20 135 20 DNA Artificial Sequence AntisenseOligonucleotide 135 gggactccaa agtcaggcca 20 136 20 DNA ArtificialSequence Antisense Oligonucleotide 136 agggactcca aagtcaggcc 20 137 20DNA Artificial Sequence Antisense Oligonucleotide 137 cagggactccaaagtcaggc 20 138 20 DNA Artificial Sequence Antisense Oligonucleotide138 tcagggactc caaagtcagg 20 139 20 DNA Artificial Sequence AntisenseOligonucleotide 139 ggtgactcag ggactccaaa 20 140 20 DNA ArtificialSequence Antisense Oligonucleotide 140 cctgactctc ggactttgaa 20 141 20DNA Artificial Sequence Antisense Oligonucleotide 141 gctgagtgagcctgactctc 20 142 20 DNA Artificial Sequence Antisense Oligonucleotide142 ccgtgctctg ggctgagtga 20 143 20 DNA Artificial Sequence AntisenseOligonucleotide 143 aaggtccctg acctgccaat 20 144 20 DNA ArtificialSequence Antisense Oligonucleotide 144 tctttcctct tgtccatcag 20 145 20DNA Artificial Sequence Antisense Oligonucleotide 145 gtctttcctcttgtccatca 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide146 ggtctttcct cttgtccatc 20 147 20 DNA Artificial Sequence AntisenseOligonucleotide 147 gggtctttcc tcttgtccat 20 148 20 DNA ArtificialSequence Antisense Oligonucleotide 148 aacagcactt tcttgatgtc 20 149 20DNA Artificial Sequence Antisense Oligonucleotide 149 ggaacctgcgcatctccaac 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide150 tggtcggccg tctggatgag 20 151 20 DNA Artificial Sequence AntisenseOligonucleotide 151 gagaagcgca gttggtcggc 20 152 20 DNA ArtificialSequence Antisense Oligonucleotide 152 aggtaggaga agcgcagttg 20 153 20DNA Artificial Sequence Antisense Oligonucleotide 153 gccaggtaggagaagcgcag 20 154 20 DNA Artificial Sequence Antisense Oligonucleotide154 agccaggtag gagaagcgca 20 155 20 DNA Artificial Sequence AntisenseOligonucleotide 155 cagccaggta ggagaagcgc 20 156 20 DNA ArtificialSequence Antisense Oligonucleotide 156 acagccaggt aggagaagcg 20 157 20DNA Artificial Sequence Antisense Oligonucleotide 157 cacagccaggtaggagaagc 20 158 20 DNA Artificial Sequence Antisense Oligonucleotide158 tcacagccag gtaggagaag 20 159 20 DNA Artificial Sequence AntisenseOligonucleotide 159 atcacagcca ggtaggagaa 20 160 20 DNA ArtificialSequence Antisense Oligonucleotide 160 gatcacagcc aggtaggaga 20 161 20DNA Artificial Sequence Antisense Oligonucleotide 161 cgatcacagccaggtaggag 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide162 tcgatcacag ccaggtagga 20 163 20 DNA Artificial Sequence AntisenseOligonucleotide 163 caccctcgat cacagccagg 20 164 20 DNA ArtificialSequence Antisense Oligonucleotide 164 tccttccact gatcctgcac 20 165 20DNA Artificial Sequence Antisense Oligonucleotide 165 ctccttccactgatcctgca 20 166 20 DNA Artificial Sequence Antisense Oligonucleotide166 gctccttcca ctgatcctgc 20 167 20 DNA Artificial Sequence AntisenseOligonucleotide 167 agctccttcc actgatcctg 20 168 20 DNA ArtificialSequence Antisense Oligonucleotide 168 aagctccttc cactgatcct 20 169 20DNA Artificial Sequence Antisense Oligonucleotide 169 aaagctccttccactgatcc 20 170 20 DNA Artificial Sequence Antisense Oligonucleotide170 gaaagctcct tccactgatc 20 171 20 DNA Artificial Sequence AntisenseOligonucleotide 171 ggaaagctcc ttccactgat 20 172 20 DNA ArtificialSequence Antisense Oligonucleotide 172 gggaaagctc cttccactga 20 173 20DNA Artificial Sequence Antisense Oligonucleotide 173 tgggaaagctccttccactg 20 174 20 DNA Artificial Sequence Antisense Oligonucleotide174 tggccgggga ggtgggggca 20 175 20 DNA Artificial Sequence AntisenseOligonucleotide 175 tgggtggccg gggaggtggg 20 176 20 DNA ArtificialSequence Antisense Oligonucleotide 176 tgcgtttggg tggccgggga 20 177 20DNA Artificial Sequence Antisense Oligonucleotide 177 tgcacttgccattgtgaggc 20 178 20 DNA Artificial Sequence Antisense Oligonucleotide178 acttcagtgt cttgactcat 20 179 20 DNA Artificial Sequence AntisenseOligonucleotide 179 aacttcagtg tcttgactca 20 180 20 DNA ArtificialSequence Antisense Oligonucleotide 180 taacttcagt gtcttgactc 20 181 20DNA Artificial Sequence Antisense Oligonucleotide 181 ctaacttcagtgtcttgact 20 182 20 DNA Artificial Sequence Antisense Oligonucleotide182 gacagatgcc tgagcacttt 20 183 20 DNA Artificial Sequence AntisenseOligonucleotide 183 gaccaggaag ggcttccagt 20 184 20 DNA ArtificialSequence Antisense Oligonucleotide 184 tgaccaggaa gggcttccag 20 185 20DNA Artificial Sequence Antisense Oligonucleotide 185 ttgaccaggaagggcttcca 20 186 20 DNA Artificial Sequence Antisense Oligonucleotide186 gttgaccagg aagggcttcc 20 187 20 DNA Artificial Sequence AntisenseOligonucleotide 187 gcacacgttg accaggaagg 20 188 20 DNA ArtificialSequence Antisense Oligonucleotide 188 gaggtacgcg ccagtcgcca 20 189 20DNA Artificial Sequence Antisense Oligonucleotide 189 tacccggtaacagaggtacg 20 190 20 DNA Artificial Sequence Antisense Oligonucleotide190 agtgaaaaca tacccggtaa 20 191 20 DNA Artificial Sequence AntisenseOligonucleotide 191 caaatcctaa cctgggcagt 20 192 20 DNA ArtificialSequence Antisense Oligonucleotide 192 ttccagttcc accacaggct 20 193 20DNA Artificial Sequence Antisense Oligonucleotide 193 ccagtgcacagatgcccctc 20 194 20 DNA Artificial Sequence Antisense Oligonucleotide194 acaggttaag gccctgagat 20 195 20 DNA Artificial Sequence AntisenseOligonucleotide 195 gcctagcatc ttttgttttc 20 196 20 DNA ArtificialSequence Antisense Oligonucleotide 196 aagccagcag gaactttaca 20 197 20DNA Artificial Sequence Antisense Oligonucleotide 197 gggacacctgagggaagcag 20 198 20 DNA Artificial Sequence Antisense Oligonucleotide198 ggtcatctgc aagatggcgg 20 199 20 DNA Artificial Sequence AntisenseOligonucleotide 199 gccaacctct gatgaccctg 20 200 20 DNA ArtificialSequence Antisense Oligonucleotide 200 tggaagcccc agctctaagc 20 201 20DNA Artificial Sequence Antisense Oligonucleotide 201 tagtaatgactttccaatca 20 202 20 DNA Artificial Sequence Antisense Oligonucleotide202 tgagtcttgc tttacacctc 20 203 20 DNA Artificial Sequence AntisenseOligonucleotide 203 cctgcgcgcg gagtgacttc 20 204 20 DNA ArtificialSequence Antisense Oligonucleotide 204 aggacgtcac tgcagcagga 20 205 20DNA Artificial Sequence Antisense Oligonucleotide 205 tcaggacaagtcttggcagt 20 206 20 DNA Artificial Sequence Antisense Oligonucleotide206 gaggctgcac agtaagcgct 20 207 20 DNA Artificial Sequence AntisenseOligonucleotide 207 tcagccaacc agcatcagag 20 208 20 DNA ArtificialSequence Antisense Oligonucleotide 208 acccacagtg tccacctccc 20 209 20DNA Artificial Sequence Antisense Oligonucleotide 209 agtgcgggctgtgctgctgg 20 210 20 DNA Artificial Sequence Antisense Oligonucleotide210 cagctcgctc tggcggcctc 20 211 20 DNA Artificial Sequence AntisenseOligonucleotide 211 aggaagggag ctgcacgtcc 20 212 20 DNA ArtificialSequence Antisense Oligonucleotide 212 ccctcacgat tgctcgtggg 20 213 20DNA Artificial Sequence Antisense Oligonucleotide 213 cagtggagcggctcctctgg 20 214 20 DNA Artificial Sequence Antisense Oligonucleotide214 caggctgaca ccttacacgg 20 215 20 DNA Artificial Sequence AntisenseOligonucleotide 215 gtcctacctc aaccctagga 20 216 20 DNA ArtificialSequence Antisense Oligonucleotide 216 ctgccccagc accagccaca 20 217 20DNA Artificial Sequence Antisense Oligonucleotide 217 attgcttctaagaccctcag 20 218 20 DNA Artificial Sequence Antisense Oligonucleotide218 ttacatgtca ccactgttgt 20 219 20 DNA Artificial Sequence AntisenseOligonucleotide 219 tacacatgtc atcagtagcc 20 220 20 DNA ArtificialSequence Antisense Oligonucleotide 220 ttttctaact cacagggaaa 20 221 20DNA Artificial Sequence Antisense Oligonucleotide 221 gtgcccgccagtgagcaggc 20 222 20 DNA Artificial Sequence Antisense Oligonucleotide222 cggcctcggc actggacagc 20 223 20 DNA Artificial Sequence AntisenseOligonucleotide 223 gtggaatgtc tgagatccag 20 224 20 DNA ArtificialSequence Antisense Oligonucleotide 224 agggcgggcc tgcttgccca 20 225 20DNA Artificial Sequence Antisense Oligonucleotide 225 cggtcctggcctgctccaga 20 226 20 DNA Artificial Sequence Antisense Oligonucleotide226 tacactgttc ccaggagggt 20 227 20 DNA Artificial Sequence AntisenseOligonucleotide 227 tggtgccagc agcgctagca 20 228 20 DNA ArtificialSequence Antisense Oligonucleotide 228 cagtctcttc agcctcaaga 20 229 20DNA Artificial Sequence Antisense Oligonucleotide 229 aagagtcatgagcaccatca 20 230 20 DNA Artificial Sequence Antisense Oligonucleotide230 tgaaggtcaa gttcccctca 20 231 20 DNA Artificial Sequence AntisenseOligonucleotide 231 ctggcaagag gcagactgga 20 232 20 DNA ArtificialSequence Antisense Oligonucleotide 232 ggctctgtgc tggcttctct 20 233 20DNA Artificial Sequence Antisense Oligonucleotide 233 gccatctcctcagcctgtgc 20 234 20 DNA Artificial Sequence Antisense Oligonucleotide234 agcgcctgct ctgaggcccc 20 235 20 DNA Artificial Sequence AntisenseOligonucleotide 235 tgctgagtaa gtattgactt 20 236 20 DNA ArtificialSequence Antisense Oligonucleotide 236 ctatggccat ttagagagag 20 237 20DNA Artificial Sequence Antisense Oligonucleotide 237 tggtttattctatggccatt 20 238 20 DNA Artificial Sequence Antisense Oligonucleotide238 cgctcctgca aaggtgctat 20 239 20 DNA Artificial Sequence AntisenseOligonucleotide 239 gttggaaacg gtgcagtcgg 20 240 20 DNA ArtificialSequence Antisense Oligonucleotide 240 atttattgtt gcaactaatg 20 241 2346DNA Mus musculus CDS (710)...(2008) 241 gaattcggga tccttttgca cattcctagttagcagtgca tactcatcag actggagatg 60 tttaatgaca tcagggaacc aaacggacaacccatagtac ccgaagacag ggtgaaccag 120 acaatcgtaa gcttgatggt gttttccctgactgggtagt tgaagcatct catgaatgtc 180 agccaaattc cgtacagttc ggtgcggatccgaacgaaac acctcctgta ccaggttccc 240 gtgtcgctct caatttcaat cagctcatctatttgtttgg gagtcttgat tttatttacc 300 gtgaagacct tctctggctg gccccgggctctcatgttgg tgtcatgaat taacttcaga 360 atcatccagg cttcatcatg ttttcccacctccagcaaga accgagggct ttctggcatg 420 aaggtgagag ccaccacaga ggagacgcatgggagcgcac agacgatgac gaagacgcgc 480 cacgtgtgga actggtaggc tgaacccatgctgaagctcc acccgtagtg gggaatgatg 540 gcccaggcat ggcggaggct agatgccgccaatcatccag aacatgcaga agccgctgct 600 ggggagcttg gggctgcggt ggtggcgggtgacgggcttc gggacgcgga gcgacgcggc 660 ctagcgcggc ggacggccgt gggaactcgggcagccgacc cgtcccgcc atg gag atg 718 Met Glu Met 1 gag aag gag ttc gaggag atc gac aag gct ggg aac tgg gcg gct att 766 Glu Lys Glu Phe Glu GluIle Asp Lys Ala Gly Asn Trp Ala Ala Ile 5 10 15 tac cag gac att cga catgaa gcc agc gac ttc cca tgc aaa gtc gcg 814 Tyr Gln Asp Ile Arg His GluAla Ser Asp Phe Pro Cys Lys Val Ala 20 25 30 35 aag ctt cct aag aac aaaaac cgg aac agg tac cga gat gtc agc cct 862 Lys Leu Pro Lys Asn Lys AsnArg Asn Arg Tyr Arg Asp Val Ser Pro 40 45 50 ttt gac cac agt cgg att aaattg cac cag gaa gat aat gac tat atc 910 Phe Asp His Ser Arg Ile Lys LeuHis Gln Glu Asp Asn Asp Tyr Ile 55 60 65 aat gcc agc ttg ata aaa atg gaagaa gcc cag agg agc tat att ctc 958 Asn Ala Ser Leu Ile Lys Met Glu GluAla Gln Arg Ser Tyr Ile Leu 70 75 80 acc cag ggc cct tta cca aac aca tgtggg cac ttc tgg gag atg gtg 1006 Thr Gln Gly Pro Leu Pro Asn Thr Cys GlyHis Phe Trp Glu Met Val 85 90 95 tgg gag cag aag agc agg ggc gtg gtc atgctc aac cgc atc atg gag 1054 Trp Glu Gln Lys Ser Arg Gly Val Val Met LeuAsn Arg Ile Met Glu 100 105 110 115 aaa ggc tcg tta aaa tgt gcc cag tattgg cca cag caa gaa gaa aag 1102 Lys Gly Ser Leu Lys Cys Ala Gln Tyr TrpPro Gln Gln Glu Glu Lys 120 125 130 gag atg gtc ttt gat gac aca ggt ttgaag ttg aca cta atc tct gaa 1150 Glu Met Val Phe Asp Asp Thr Gly Leu LysLeu Thr Leu Ile Ser Glu 135 140 145 gat gtc aag tca tat tac aca gta cgacag ttg gag ttg gaa aac ctg 1198 Asp Val Lys Ser Tyr Tyr Thr Val Arg GlnLeu Glu Leu Glu Asn Leu 150 155 160 act acc aag gag act cga gag atc ctgcat ttc cac tac acc aca tgg 1246 Thr Thr Lys Glu Thr Arg Glu Ile Leu HisPhe His Tyr Thr Thr Trp 165 170 175 cct gac ttt gga gtc ccc gag tca ccggct tct ttc ctc aat ttc ctt 1294 Pro Asp Phe Gly Val Pro Glu Ser Pro AlaSer Phe Leu Asn Phe Leu 180 185 190 195 ttc aaa gtc cga gag tca ggc tcactc agc ctg gag cat ggc ccc att 1342 Phe Lys Val Arg Glu Ser Gly Ser LeuSer Leu Glu His Gly Pro Ile 200 205 210 gtg gtc cac tgc agc gcc ggc atcggg agg tca ggg acc ttc tgt ctg 1390 Val Val His Cys Ser Ala Gly Ile GlyArg Ser Gly Thr Phe Cys Leu 215 220 225 gct gac acc tgc ctc tta ctg atggac aag agg aaa gac cca tct tcc 1438 Ala Asp Thr Cys Leu Leu Leu Met AspLys Arg Lys Asp Pro Ser Ser 230 235 240 gtg gac atc aag aaa gta ctg ctggag atg cgc agg ttc cgc atg ggg 1486 Val Asp Ile Lys Lys Val Leu Leu GluMet Arg Arg Phe Arg Met Gly 245 250 255 ctc atc cag act gcc gac cag ctgcgc ttc tcc tac ctg gct gtc atc 1534 Leu Ile Gln Thr Ala Asp Gln Leu ArgPhe Ser Tyr Leu Ala Val Ile 260 265 270 275 gag ggc gcc aag ttc atc atgggc gac tcg tca gtg cag gat cag tgg 1582 Glu Gly Ala Lys Phe Ile Met GlyAsp Ser Ser Val Gln Asp Gln Trp 280 285 290 aag gag ctc tcc cgg gag gatcta gac ctt cca ccc gag cac gtg ccc 1630 Lys Glu Leu Ser Arg Glu Asp LeuAsp Leu Pro Pro Glu His Val Pro 295 300 305 cca cct ccc cgg cca ccc aaacgc aca ctg gag cct cac aac ggg aag 1678 Pro Pro Pro Arg Pro Pro Lys ArgThr Leu Glu Pro His Asn Gly Lys 310 315 320 tgc aag gag ctc ttc tcc agccac cag tgg gtg agc gag gag acc tgt 1726 Cys Lys Glu Leu Phe Ser Ser HisGln Trp Val Ser Glu Glu Thr Cys 325 330 335 ggg gat gaa gac agc ctg gccaga gag gaa ggc aga gcc cag tca agt 1774 Gly Asp Glu Asp Ser Leu Ala ArgGlu Glu Gly Arg Ala Gln Ser Ser 340 345 350 355 gcc atg cac agc gtg agcagc atg agt cca gac act gaa gtt agg aga 1822 Ala Met His Ser Val Ser SerMet Ser Pro Asp Thr Glu Val Arg Arg 360 365 370 cgg atg gtg ggt gga ggtctt caa agt gct cag gcg tct gtc ccc acc 1870 Arg Met Val Gly Gly Gly LeuGln Ser Ala Gln Ala Ser Val Pro Thr 375 380 385 gag gaa gag ctg tcc tccact gag gag gaa cac aag gca cat tgg cca 1918 Glu Glu Glu Leu Ser Ser ThrGlu Glu Glu His Lys Ala His Trp Pro 390 395 400 agt cac tgg aag ccc ttcctg gtc aat gtg tgc atg gcc acg ctc ctg 1966 Ser His Trp Lys Pro Phe LeuVal Asn Val Cys Met Ala Thr Leu Leu 405 410 415 gcc acc ggc gcg tac ttgtgc tac cgg gtg tgt ttt cac tga 2008 Ala Thr Gly Ala Tyr Leu Cys Tyr ArgVal Cys Phe His * 420 425 430 cagactggga ggcactgcca ctgcccagcttaggatgcgg tctgcggcgt ctgacctggt 2068 gtagagggaa caacaactcg caagcctgctctggaactgg aagggcctgc cccaggaggg 2128 tattagtgca ctgggctttg aaggagcccctggtcccacg aacagagtct aatctcaggg 2188 ccttaacctg ttcaggagaa gtagaggaaatgccaaatac tcttcttgct ctcacctcac 2248 tcctcccctt tctctgattc atttgtttttggaaaaaaaa aaaaaaagaa ttacaacaca 2308 ttgttgtttt taacatttat aaaggcaggcccgaattc 2346 242 20 DNA Artificial Sequence unsure (1)..(20) AntisenseOligonucleotide 242 nnnnnnnnnn nnnnnnnnnn 20 243 75899 DNA Homo sapiens243 gatcttcctg cctcagcctc cccagcagct gggccccacc acaccggcta attttttaac 60ttttagtagt gacgaggtct gattctgtta cccaggctgg tctggaactc ctggcctcaa 120gacatccgcc tgcctctgcc tcccaaagtg ctgggattac agatgtaagc caccgcgcct 180gggctcctat gatttttatt taacataatg caccatggaa tttgtgctct gcttagttca 240gtctgagcag gagttccttg atacttcggg aaacactgaa aatcattcca tccccatcca 300ttcattcctg cagcacccaa gtggaaattc tgcgtttcag acagggacac tacccttaga 360gagcagtggg cttccccagc agcgtagtga aacatgatac tcctgagttt catgaaaaaa 420gggcagacat ctggccagag ctgggaggca ggaaatagag cacggtgccc tcctcccata 480ctccagcttg gattactgag gctggggccc aggccctgca ggaaaggagg tgcatgacta 540ctttaaggcc actcactctg tgactcaacg ggccgggtcg gggctggaac tcaatgccct 600cccgggcctg gagagcccac gcgccgtggg cggggctccc ggggtcgcct aggcaacagg 660cgcgcgccgc gcccgagccc agagccccaa agcggaggag ggaacgcgcg ctattagata 720tctcgcggtg ctggggccac ttcccctagc accgcccccg gctcctcccc gcggaagtgc 780ttgtcgaaat tctcgatcgc tgattggtcc ttctgcttca ggggcggagc ccctggcagg 840cgtgatgcgt agttccggct gccggttgac atgaagaagc agcagcggct agggcggcgg 900tagctgcagg ggtcggggat tgcagcgggc ctcggggcta agagcgcgac gcggcctaga 960gcggcagacg gcgcagtggg ccgagaagga ggcgcagcag ccgccctggc ccgtcatgga 1020gatggaaaag gagttcgagc agatcgacaa gtccgggagc tgggcggcca tttaccaggt 1080gcgggagcgc cccggagcgt ggcgggccct tcgcttaggc cgcttgaaca tcccctcaga 1140cctccaggcc ccagactccc tctgggtctt gccctctgcc tcgctcctac tgcttgagga 1200ttcgatggga cagcgacgca ctgcgtcccc ccaccctttg tccccggggc gggcgtgttt 1260ctcgccgcag cgtcggagcc cccttcgatc ccccacctcc cttctgttct ccagctcggg 1320tgatctctca agccggggga ccgccggtct gtgctctcaa cgcgaatccc tcgcaccccg 1380accccgcccc ctgcctgtcc actctttgtc ccctggggtg atttagcacc cccactattt 1440ccttttctgg agtggaccac ctcagactct cttcctttgt ctccctgggg gaaaaggtta 1500ctccccccgt ccctccttca catttccttt cccctagtct cagtgtgcgt cgagtcccag 1560agatgacagt cccctttccc ctttctgttc attcatttat tggataggag ttggcaagct 1620tattctgtgc taggcaccgc ttaggcattg gaggtggtgt ttgctaatca ggacaggcaa 1680gatcctagcc ttagtggggc ctagagtcga atagggcaat caaacacaaa agcaaataat 1740ttcagatagt gacaggtgct gtgaagagaa cgacttccta acggggtaca gggtgactgc 1800atagaaggcc ggctgtctta gagaagggga tcagggaagg cctgtcaaag gaggagacat 1860ttgctttgtg agctgaacca agaggagcag aaagccgtga gaatatgggg ctaaagaacc 1920ttctagccag gaggcctgcg gtacccactc cattggggcc atgatattat tctttcaggc 1980agggactcag gaaggttaac gttttaaccc tctctaaaat agcatctttc ctcaatgagc 2040agcttagtct ttggtcgtgg cagagatgac cttgtcttag gagtcatctc cttgtgtgtt 2100aaaaagttag gaaaggaggg tttctcatat atctataaaa caagtagtta aaaacacaaa 2160gagctcttcc tttcacaagc agctgaataa gatacatact cccaattaaa tgtcattgcg 2220ggggttgtta agattaacta aaaccacact tgcacagtat cttaaataag cgatatacag 2280aatagagaga ttttgttact tgtgtaaagg gagacagcag atgattctgt tttcagctta 2340taggctcaaa aggcaaattg tgagatccat cagctgtagt attaaaatct attttgagct 2400ccgcttagaa aggaaaaaag gtttaagcag ttctttggta tgcttgacta acaaaagcct 2460ttttttttgg cagccttgat tttcatgtgg atttacatca agcttatttg acaggattct 2520ttttatttgg actgtagtgt gtatattagt ttctgctaga ctaatatttc taaccactgt 2580aatctatata ctaataagta tgattgatca gtatataaaa tttgtatgcc atatctggtc 2640tctgaattag ctgaatgaat tccataaggg actttgagac tgtgtagaca aattttctgc 2700atcagtttaa tgcagtagag tctaaaatgt ctttaaatga aaattgttgg tctgaagtgt 2760tggagttgat tatgatacac cccatcacag tggaagcatt gtggagagaa gtcttttcca 2820ctgaaattga ctgagttgac aacaagaaat acgtattgta acttagttct tagttgaatt 2880ttatttctta caattttaag ccagagtggg ttgacctgtc acccaagcat ggttaaaatt 2940gtattcagca tgcaactagc atggagtgtg tcagtcttca attcatttcc ttcattgttc 3000ttaagttttt ctgccacaat taaaccccac aagttagtca aggtgttgag attttcactg 3060cttcttaatg gattgccaca ttccctgagg tagtttcttt tggtcttaga gaattgtcag 3120ggccagcttt tctcacctcc actgtatgga tatttttctt ttctaagatc ttgaaatcag 3180aagcttttct cctaagtgta aaagtagctc tttgtcatac aactgtagcg ttttctgaaa 3240cagagttcag atgaccttga gtctaaagtg gctaactttc caaggtgtgt atcgctttac 3300caaaaccatt atttttcaag gattcaaaga atgtgtttac aattgataga aaatggaagt 3360ttaaaaaaat taatacttta tagcatgttg aaatgagggc agccttatac aaagtcatac 3420tttgagcttg cctagcctat tgtgatcaga gaataatgta atttttgctt acaacttggt 3480aagcaggtca gttattctaa cttattttct gattagaaca aaaagatgta aaaacttgaa 3540aactattggg aaaagaacaa agagtgaaga ggacttttga gtgctgagga atgtggcagc 3600ttggaaaaca aactttttag gcagagattc tttgctaggt cagtttgata aagtgagcat 3660aaccgtattt ttaatcttta atgctaatga atagcataga tgctaataag catctaggtc 3720tataaaaagt cagctttgat agtgtatata gatggcttta aacattgttt tctagcattt 3780aaacactttc aaatcatccg gttgcttgat tgggcctagc tgtctaagag gagagaatga 3840gcccagatga ggaaaagaga ttgattttac tgagctagaa tgagaggaga gagggttgag 3900tgaatgaaaa gaatagctca tgtgctcccc tccatctgta gtttaagagg ggttgggtcc 3960ggtgttttgc ttgttttctc gtctgtaaat tctttgattc tctgacacca ctcactatat 4020ttcattgtga atgatttgat tgtttcagat aaaggggact gcaataatac cttgtgacat 4080gaaggcaaga tttattcatg ttagaggcag gctttgtaaa atgggccact cttccaattg 4140acatttgttt ttatagctgt tttcattatg aaatacaatc taatgcctga ctaggttaaa 4200accatgttgt aacaatagtt cactaaaatt ccttactgat atacagctta tgttgttata 4260ttccaaaaag atgaatatta aaatttgcca ataatgttta tttaaatact attttcttca 4320gaggaaaaaa aactatttta tgcaaaggag aaagatctat acactatgac tcacttcact 4380taaaaaaaaa aagactaacg gaaatgacat ggagagactg ggaagttcta gtcatcttga 4440gtgacccatt agatctaaat gttcttgttt agccctggtt tgagtgaact aaatttaggt 4500gtctgatcag tactttggaa atggtgtaaa tgcctttgta attgtctgga ctgatattag 4560attaactggg agcacaagta gaaatagtga aggaaagaac tttttgctat tgttatttga 4620catcactggc atatttatag gaatactttg gtgtttttgg aagtaagtaa accaaccagt 4680ggttctaaaa agtcagctgg gggataatgg taatgccgct gtttcttagc tgcaagttat 4740ctgccgttac ttctcctcca ttttgcattt tatcttgaat agctcctcaa aacctattaa 4800aatacctggt attgaataat gtaattgaat gtgtactgaa tttcacagtg gaaatgaata 4860agaaatttcc tgtggaggtt ttttgactta gctactgaaa taacggcctt ttgttgtgtg 4920attctttccc ttttctcttt gttaaagaaa actgtcttgt gatcttgtag attacagaat 4980ccttttggca atttctgttc ctagcactgc tttttctttc tttctttctt ttaaatagaa 5040atggggtttt gctgtgttgc ccaggttggt cttgaactcc tggcttcaag cgatcctccc 5100accttggcct cctgaagttg ggattgcagg cgtgagcagg tactttttct gaggcctgcc 5160tgagcctata tatattttgc acaatttggc attcctccct acagtgttta tgctgatttg 5220tttctggtaa caactaatac tggcaaatcg gctgggcatg ttactttatg ctgcccatat 5280tcaggaaaat tggaattcta gctgggtcat tgttcccaga tgatgtagtt tggcaccagc 5340cattccatgt tcacattttg agtatccagg agggctgggg actttggagt agttggtgat 5400tccctctgcc acatttcact ggttggtcac tatggcatcc tttccaccac actagtagtc 5460taggttctca gatgttgctt atgagcctgc aatggtttct agtttcacac tgcagaaatg 5520agtgaagccg gttacccgtt aatatggtcc catcatcact agagtaattc attgttctaa 5580aaccagatct gagtctctca ctcctctgca actacttctg attctttcat aacacttgta 5640aagtccaaac tcctctttag catggcagcc agcttccagt ccttccctcc tatgtggctt 5700ccattctagc cagacaagaa agggcagcgt tctccaaact catcctcgcc cttcattcct 5760ctataccatt gctgagcact ttgttgagga tgcctctccc gttcaatcta gcttgcatct 5820tccagctcga atgtgtgctt ccttgcacca gagttttgtt ccgtcacctg tgtgttttca 5880tacaagctgg cacatatctc ttctaaagcc ctgctgtcat tgtagctgcg tctttacaaa 5940catttttttt ttaaattttt ataaagtcaa ggtctcacta tattgcccag gctggtctca 6000aactcctggg ctcaagtgat cctcctgcct tggcctccca gagtgctggg attataggta 6060tgagacactg tgcccagctg tagctgctac tttatatccc aggtctatct ccaatggagc 6120ccaagcttcc tgaggccacc tgttgtatct ttctcattca tcttgaagtc ctctgctcct 6180ggcacagagt aggtacctaa caagagttgg gattgaattg atggtcagta ctttgctagc 6240ctgatggtat aaagatgtac aaaacatgtt cctggctccc actctagggg ggcaatgatg 6300gaaacaaata gattagccca cattagtacc aatagtagag gtcactctgg gagaaggccc 6360ccaccacatt ttgagtcatg gcctaatgag gtaatttagt attgcctgct gcagtggctt 6420tggaagaaag gctggcattc ttagccagta gaagctgata ccactgattt gtttcacaga 6480agctttaaat ataacaataa atttgtgctt ggcctacggt gaactttaca ggcaacttgg 6540aggtaatatg tttgtctctc taagaattgt tgaattcctc ttccctcatc cctcctgact 6600ggttctcaca agcctagcgg gcctttgcat gtggttggtt cataaaatac tttttgattt 6660tgggatataa aatatagttc tccataaaat aacgactgtt accaagtctt tgattttttt 6720tttcaaacta taaatggtaa tgacattctt tggcctttga tcagaccacc cttaggggca 6780agagagtagt ttcatgtttt gctttttcta gtgtcccctg tgtctgggta tagttgcagt 6840ctcagctgtc atactaacag tgctgagtga gtcccttact ttctttgggt tttggtttct 6900cccttgtaaa aatgatcctg gactaactga tcattaagtt caggtcaagt aataaaaatc 6960cttaatgtac tcacaaatac aatttaatgt tcctgaataa tccttgtaaa aactgcagca 7020gttactcagt tttgtaaggt gtggttgggt actattaggc tcaaaagttt ataggagctt 7080tgtgagtata gttaacaact caaaagaatg gggtgttttt tcccgagggg catgaaatgt 7140ttttgataaa tagagttcat ttgacttggt aatgtggaaa atgagtagcc ctgacacgta 7200cgctatgctt ttgcagtttt tctctcaagt agcaattggg tggcttttcc tgtaaaagat 7260agaggaactg attcttgaga atttacgaaa gcttcaaccc taactaggta tgcaaagaat 7320agttgccctt tatgttgtaa ttttaggaag aaacctacat ctggtctaag tttcatttga 7380ataatatgat agtttacaca tctgccatat ttgagaagaa agtacctaag tctccagcat 7440tttagaaata atgctttact ttgtgtagaa atggtcttta gagtttaata gctgctgccc 7500tctccttttt caaagcagct tgacataatc atgagtatct tgctgacagc ttgtaaattt 7560tgattgtatg aaaactgaaa ataagaccat ttcacatgga agattccctc ctgccctgaa 7620acagccaaag aaaactgtag ccatcaaatc tattgatctc tgggctttgg tacaagtcac 7680actactacaa ataaaataat accaagtact tataaatgat tttcagtcct tttaaagttt 7740atttttttaa tatttttttt gagatggggt cttgctgtgt cgtccaggct ggagtgcagt 7800ggcacaatct tggctcactg caacctccac ctcctgggct caagtgatcc tcccacctca 7860ggctcccaag tagctgagac tacaggcatg tgccatcacg cccagctaat ttttgtattt 7920ttttggagta gagatgggat tttgctgtgt tgcccaggct ggtcttgaac tcctgggctt 7980aagccatctg tctgcctcag gctcccaaag tgttgggatt acaggtgtga gccactgtgc 8040ccggcccagc ccttttttta agagaaaaac gtatgacatc gttcgattta ctgagtgctt 8100atggttttac taaggcagta aggttttatg gataccctat ggtaattaga tagaattagt 8160gctctgaagt cagctctgta atatggactc agagtaaaca tggcaaaggg acacttaagg 8220tctgcatttt ctctgggaaa taaacgtatt ctttactact ctgaatctag tgctgggaaa 8280ttctaaatcc ttcttgagga ttaaccactt gaagtaaagt tttgggtccc aagtaggctt 8340gtgtccctgt ctccttctct ttacttttca gatgtttctt cctagagact gaggtatatt 8400ttacttttac agatgaagaa ggaagcctcg gctgtgtttg tggcttttgt gggtgagcaa 8460catcacttgc aaagataaga tgagcatagc aaaactaggc tttcaaaata atttttaaaa 8520atttcttagt gattagaaaa ggaaaactct tcccttgtct ctgttaagaa acgtttttcg 8580acttttttcc tttcttaatg gatcttttat tggcacttct cttccttttg cagaatctta 8640cttaaaagtc actacgttac attacagcaa acagcttagc taatttttat ccagatgggc 8700cccggttaca ggattgtaca ctattgcgaa tttcttacag gaaagtgaac atcaagtaat 8760tattccaaat agagttctct taagaacgtg agttacttaa aaatgtctaa ggatgaagtc 8820acttctgaat ataacttcac tcaagagaac aaataagcaa actgcattta gcataacatg 8880gtaaattagc tttaactctc cttgatgttt gaacatttgt cgctgttaac tactgtttca 8940cttttcaaat agtcagggct tagtttgctt ctgtaaggat aaagggaaaa tacgccttca 9000ctgagtcata aatatttttg tggctaactt ttgcacagag aaaagaggcc tctaagaagg 9060tacccagtga attttttttt cggggcaggg agagaatatg tcattttttg gtttgttgtt 9120gttgttgtca ttgttttgct ttgttgtttt tactctgaac tgaactgtat cttgacagca 9180cttttgaatt aagagcatta ctcttattgt tctctactac ctggacgcca cctccctgtt 9240gccatagtgt taaggatcat gctccgaggt ggggtgaggc agaatggggc caagatcaga 9300aagttacatt aagctacatc aggtttatac aagcataaaa ccaaattttt ggagcagtcc 9360ccagaataca acctggttta gccacaccta aaggttgctc ttgaatattc cttgagaatc 9420cacatcccta gaatgctggg tttcaatggg ccctttatgt acctatcatg gtgtcatttc 9480tgagcatttc taaatattcc ttcatgtctt actgacagtt tttcttgaat aaatcttagg 9540aatattagtg ccattatcag tattttgttt ggtctgttca caccacaaat aactacccag 9600gtctgctact tgcccctatt tctctacctg ctaatgaaaa tgcttttgaa agtttgagta 9660acagtattgg agtgtgcaca gtggtattgg taggttctgt actcatcctt aaccacttgt 9720tttcatcctt tgtgagcttg aagtttctcc aaaaaattta tcacaaaact tatcagacat 9780agttaataca ctcagagaga gaatcactga aaaagtagat gtagtttaac aaacccagtg 9840cctttttttt acccatgaat acatatttgt caactaaacc tcattttgca acttgttcca 9900ctactcgaat ggtaacaaac ttttggtttc ccaatagatt tggaagatgt tgcttttgaa 9960agtaggaaat agatggcttt agaagatgga agaatatttt gtttgaagtg ggagcgtggt 10020atgtccttag ctgtctgtga aatgcagctg aagatgggtg tgggccttca tctgcatttc 10080ccatcttcag tttgaggagg tagttaccct tctaaccact taagaactgc atggtacatg 10140ctgttttatt tacagggcaa aactgtgctc ccgtagtttc cctggtgctt gccttcacgt 10200taacacagtg tcatcgtttg gcagtgttta tgtgccaggg tccatgttag aaggaggaaa 10260ggtatagcga agttaaaggg tgcagttggc ctcccacctt tagttttgta agtgccttta 10320aagtttgatt tttgtaggtt gatcataagg aagtgataag tatgttaggt tatttgtggt 10380ttgagctaat tttagtctct ttttacagct tgctttgtat cctttgccat taaaacatgc 10440tttctagaaa gacaactttt gaatgtagga cacagtctat attctatact tggctacatt 10500tcaaaaaata ttttctcagt actttggaag ttggacagtt ggaagcatag tgacagtatt 10560taaaaatctt tgattccggc cgggcatggt ggctcacgcc tgtaatccca gcactttggg 10620aggccgaggt gggtggatca cttgaggtcc ggagttcagg accagcctga ccaacatggt 10680gaaaccctgt ctctactaaa aatacaaaat tagccgagcg tggtggtaca tgcctgtaat 10740cccagctact caggaggctg aggcaggaga atcgcttgaa tctgggaggc ggaggttgca 10800ttgagccgag atcataccat tgcactacag cctgggggac aagagtgaaa ctctgtctca 10860aaaaaaaaaa aaaaattaag tgatttcttt gctttgtgac acttctactt ttccagcaag 10920taaattatat tctttcatac aggtatgaaa ttcttgttcc aagctagtgg ttaaaaaggc 10980acagttgata ttagaggatt tgtaaaagat tatgaccacg cctgcaatgt actgaagcaa 11040ggctttgctg ggctgtgtat aggaaacctt ccccagcctg tgcccttgct tgatagaaca 11100ttttgctcct aagggtaggt gcctgtatct gtctccagta ctggttagtt tcacacagaa 11160cagttgtgtt tcagagcttt agtctcaagc tgccctgctc ccctgaagca gccaccctga 11220gcatgtgcac tcacaggagg ggacatgtga ggtcatggaa gaagacgact caggaagaag 11280aagacttggg tttgggttct gactctgcct ttgactgttg tgggattttg aggagttgca 11340tacaggatct gtaaaatgta gtcattagac tagactagac agccatatag cattacctag 11400atgtaacttt ctacaaagac atggtcacag gagaagacca gagggtgggg tgatctttct 11460ggaaaaattg gggcttcatg ccttactcat gctagatatg gtagcattat atggctgtgc 11520ctgatccccc taatctaaaa gtgggacaga actttaaaat ttcatattaa ctcaaattaa 11580aacttgaaaa aaacccatta tttccttaaa aataataaaa tgccctgtgg gggcataagt 11640cacattatat tttaaaattc ctgaatgcca catggatgaa tgtagttcct tttgaaattc 11700ttcttttgtc taaagaggaa tgttggattt tgtaattgga ctaaaaaatc ttccatttga 11760gagagaaaca gtctgctgca tgttctaccc ttgttcagga taaaacccac taatagctaa 11820catttattga attctgtgtt gtgcctcagg cactgtgcaa agtcctttac atgcaatgct 11880gtttattata tactgtcaat tggtctataa cagcaggaaa tgtttcagga ggacaatgag 11940gtcccagacc ctcagtcttc tcctgtgtcc tggattcagc ttcacaatag cactatggca 12000gtgtggccac tgcttcagct tccacataca tggctgtgaa gagagacagg ggattgtgct 12060aagcctcccc gatttattag gacataggag gagagagttt gtagtttttg acctttgcct 12120agttttctaa cctctttcct agatgtcaca aattggccac ccacagtcat attttgcttg 12180cttcacgcaa tgctttttaa aaaagagaag agtttaattt gtgccattgt ttataaatga 12240atcaggagaa atgacatgca actctggatt ctggcctctc ttgaaaaatc tgaaaatcac 12300accgtctgag cttacactgg cagtggtctg ctggactgag ggacacaact ccttttggat 12360gtacatgtgt gcgttgcaga gtttaccaca gtcccacagt gggtcacact gtccttgtcg 12420gtgtacacta cctagcactt gagtttgcaa cccctacccc aagctgagtt ttctcgtcaa 12480gcttgatgtt aatgttatgt gatgcttggc cttgtaggta tttggtatat tatcgttaga 12540taaaattgaa gcaaagggct aaagggttgg tggcctgagg gagtgccctt gacagtaaag 12600tctaggataa aatcattggc caggtactcc ttcccttccc gcccttcctc ttttctcttt 12660atcctcagcc tccttctgct attttgagga agttagaagc caccaccatt ttttcccacc 12720tcaggcaact gagtgtggct gtatttctgt cccatgttca gttatttcca ggaactattt 12780ttgatgacca acttgaagtt acattgggtg ggcctaatgg gggctgataa aagaatgagg 12840tgaccaaata tgcttgcact gagacggcta cgaagtaagg tttttaatga cttgctttgt 12900gacttggtca ggagtgatac catttgtcat gtgtccaact tcatgactaa atggttgctc 12960taccttatcc tcatagctat aataaaataa aataaataca tacattgcag ggaggaatgt 13020atcttgttaa aggtctctcc cttttagcaa caaaagtaca tattatgttg tagaacatgc 13080tttttctttg atccttcttg aacacctatt actctataga ggtatgttgt gtatggcaaa 13140ttagaacaag caatagataa ggatgattct ttaccattat aacccagtca aggtctttgt 13200cctaagtttt gtacctttct ccagagggaa aggtatttgt atttatttat ttatttttga 13260ggcagagttt tgctcttgtt gcccaggctg gggtgcaatg gcacgatctc agctcactgt 13320aacatccgcc tcccgagttc aagtgattct cctgcctcag cctcccgagt agctgggatt 13380acaggtgcct gccacgatgc ccggctaatt tttttttttt tttttgtatt tttagtagag 13440atggggtttc atcatgttgg ccaggctggt cttgaactcc tgacctcagg tgatccatcc 13500acctcggcct cccaaagtgt tgggattaca ggcatcagcc actgcctccg gccaggtatt 13560tgtattttta gtctctatgc cttaccgtct cagatcagga ggatttggtg atttatcgaa 13620tgtgggggaa ggggaagaag aggaaacggg aggaatgttc cagattaggg aaatagctag 13680atggaagatg cagcccctca tcaaggtggg gacacaggaa aaggaacgtg tgcaaagaag 13740atggtgatct ggttgtgacc atgttgttag aggacgtcca gggaagcatc tggtaggtgg 13800tggggtgttt aaatatagaa cattcggaga atgctccgaa gcttcagaga acccttccca 13860aaaggacaaa accagctcag tgttttagca ctccgggatc atatggcatg acagcatggc 13920tgctttatac ttttttgtgt atgtgaaatt aaaaccaacc actcaggacc aatttctctg 13980aagctttttg tcaatctttc atttgctttt ctcgtctaga ttgtaagctc cttgcagcca 14040gtgtctgttg attcagtcat tcaaaaaata atacatgaac agctactagg taccaggctc 14100tgtgctgggc agttgggata tgtggtgagg aagacaaact tggtccctgc ccttaggaag 14160ttcagtagtc cagcagacaa agtggctgaa taaagataat ctcagttcac agtgataaga 14220gctcttacag gcctaggctc caggtgctgt ggggatgctc aggaaaaggt atctaattgg 14280gattgggagc aggcaaaaca aataaaggat agtgtataaa ggtaatatct agttgaagtt 14340ctgaagggca aggaggagtg agcctgtata ttctctgagt ctctccctaa tctgggattg 14400acttcttgtc cgtctctgtt catattaagt gtcacctagg cttgaaaggg tgagatcata 14460tttcacttcc ttcctctttg gtcttaacct ttctctgcta ccccctcaca caatgcatat 14520gcattattct cttattgtat atatttttcc tctcttcctt ttcatgtttc ctctgccatt 14580acttttaacc tcgactgcca tatggcctct aaacgcttcc agaagggtag cctagtggag 14640gttattccat catggccttg agctcatgcg accagatagt gaaggcatct gtgtaggtgt 14700cttctccagg agggtgatat ttgtttcatt gtaaattttg tagccctaga acaccaacaa 14760cagtgcacag taattagtag gcaggcagta caggattcat tgaagtgaag tgataacttt 14820tatccaagta tgtatgcaga taatctttga tttgtacaaa aaaaattata ttttaatatg 14880taaagatttt ttaaaagaat cttcaagttt tagccttccc actaggaata tattgaaaac 14940atgtgcctag ttcactgact tgcagctgcc actatgagaa taaaggtctc atttagttgt 15000tgtgaatttt aagggatatt ttcaatgatg ttggctggtt tatcccatta tgtggtcttt 15060tttttttttt tttttttttt ttgaggtgga gtctcgctct gtcacccagg ctggagtgca 15120gtggcgcaat ctcgactcac tgcaacctcc gcctcccggg ttcaagcgat tctgctgtct 15180cagcctccta agtagctggg attacaggcg cctgccacta cgcccagcta atttttggta 15240tttttggtag agaagggttt caccatgttg gtcaggctgg tctcgaactc ctgacctcat 15300gatccactca cttcagcctc ccaaagtgct gggattacag gcgtgagcca ccatgcccag 15360cctatgtgct cttattagca attctcagta cacagatagc tttgagtgat tctttcaagt 15420caagtacctt attaaaaaac tcaagtgtac tgataattat cttactttta aatggctaag 15480tgataagact gaatttttag gtactgtaac acttcagatt acagattctg atatttttat 15540ggttatttat atttatttat ttttgagatg gagttttgct cttgctgcct aggctggagt 15600gcaatggcac gatctcggct cactgcaacc tccgcctccc aggttcaagc gattctcctg 15660cctcagcctc ctgagtagct gggattacag tcacccgcca ctacagccgg ctaatttttg 15720ttatttttaa tagagacaat gtttcaccat gttggccagg gtggtctcgc acttctgacc 15780tctggcgatc cgcccgcctc ggcctcccaa agtgctggga ttacaggcgt gagccaccgc 15840acctggcctg gttacttaaa tttaaataca aaaattatgt tgattaattc tgaatgattt 15900cctgattgct ccccgtttac cattcacaca tttattaaat tcttcgcttg ccatatagaa 15960gcagtctctc tgccatatat gccatataga taacagaact agctgtctgc aaaccactga 16020aattgtgaaa acatctcccc ttttttcctg tttctaattc tagctatgag gattatatac 16080agaagtagtc ctggatttga tttttttttt tttttgatga ttgttttttg atagttgttg 16140actacaaatc atttaaacgt ctgaaagggg aaaggttttc cttaaaaatg gatgacaaag 16200gagaataaaa aggtattttg actatttttt tgaatgatga gttttttttt tctctttctt 16260gttttctttt ggagtcattt atgtgtcact gagtggatac catggaacat gtggcagaag 16320tagatatatg gggtaaaaga accatagttc ataagctcct tgacagaatc actgaagtgt 16380agccgttata tggccactgt cgcaggggga ggcagcagtt ttgaagaagg ggatgagtaa 16440taatgagtga taaaaaggca tcctggatag aagaccaaac tctgcagaag accccagttt 16500gattatgctt ttgttttctg atttgcggag gagagtgaaa atgcctgagg ggtgcggggg 16560agcacatagg gtgtatgtgt gtgtgtgtgc gcgtgcagat tctctctttc actgtatgta 16620tttgtatgca tgtatgtatc ttaggactta agctttctag tcaataaatt gccatagtgg 16680ggaattgctt aattgcttgc cttctgttgt tgtatttaat ttaattttat ttttaatgat 16740ttttttggtg gggtacaggg tcttaactat gttgtccagg ctggtcttga actcctaaac 16800tcaagtgatc ctcccgcctc gggctcccaa aatgctggga ttacaggtgt gagccaccat 16860gcccagctta gttgtatttt aaatgggcct gtttgcagca ttccctactc cccttagttt 16920acctggctca caacctgtct ttccatatca aggcttctgt cacccctggc ccatgtcagt 16980gcatttgggc agcccaccca gcatcatcac ctcatgtccc agggaacttc ctgttcctct 17040cttccagcta tttccttccc tggcagttga gatagtctct acctttgacc tactgttaag 17100ctcagacctt ctgctctcta gttacagcct ctgtgctgcc agattccctc gctcagttgc 17160tttctctagt ttgggttttc tcctttattc agatttccag ctgtttctct cctcccccca 17220ccgcagcctc ctcacttccc tccttatgca tctgagactg tggtcagtca ctttagatgc 17280tgcctctcca ctgtacttgt gtccatcttc ttacctacca cctctagccc tggagcaggc 17340tcttcccctg tctttgtctt cctgggccca ggctcctaag cgctgctgga aaaaaaatcc 17400cccagtattg agcccctaga aatccagtct ttaatcccaa atctgtctcc cccagcatct 17460ggccatcaga tctaaagctt acctgccatc ctttccacct catttctctc acaggggaaa 17520aggagccttt gctcctagag tctgcgctcc tgaccccttc ccatctcacc tgttcaaggc 17580atcttgcaat aaggggttgg tgactctcga ggaatggatc ccaggccctc cctattatca 17640tcttatgtat gccagttcaa cgttctcagc ttcctccagc cgagacggcc cctccagcca 17700ctgctttata ctctccttct ctggttgaaa tttttgaagt aaataggtca ctctgcccat 17760cgttcatctt ccagtcactc tgtgtgttta tcttccaggg aagtgaggct ctatgctacc 17820aagccactga aataattttt ttttttttcc agactgagtc ttgctctgtc acccaggctg 17880gagtgcagtg ccgcagtctt ggctcactgc aacctctgcc tcccggcttc aggcgattct 17940cctgccccag cctcctgagt agctgggatt acaggtgcct gtcatcacgc ctggctaatt 18000ttttgtattt ttggtagaga tggggcttca ccatgttggc caggcttgtt ggcatgttga 18060ccatgttggc caggctagcc tcaagtgatc cacccgtcag cctcccaaag tgctgagatt 18120acaggtgtga gccaccgcac ctggcctgaa ataattcttg acaagatctg cttccttgtt 18180actaatacag tggatatttt gcatcctaat tttaatgcag ttcagtgtgg tagacctgta 18240tttgcatatt gaatattccc ttccctgttt taataactct attttttcct tttcttttat 18300atctcctgct tctctagcta gtcctagacc ttactcatcg gtgtcttctc tgtttgttcc 18360tcaacttgag gagttcctac agggtttacc caatctgctg ctttcattta gcccttttgt 18420tctttttgag ccatctcatt cactcaccca ggatgtagca tcggcccttg aattcagtgt 18480gcacacatac actgtgcact atgggacagc cttcagaggc actttgttcc tgaaattgtg 18540gtggtctttg cctctcatgg agccttgcat atgctgtttc ctctgcctgg aatatcctac 18600cttttactta actgattctc gttcttcttt ccagtcacat tttgtacatt tcttctggga 18660agctttctct gatttcccct ttccacaggt ccaagttaac tgccttgtct aggtcctccc 18720atggccctct gaaggcctcc tttcatagca ccatgtctga gtatactgta ataacacgca 18780ttgctctgta atagcctgtt tacttaccta ttgccaagta atctatcaag tcttataaag 18840ggcggggctg cttttgttct agtcatttgt atctcttagt acccaatata gtgtttggca 18900tatagaaaat acccaacaag gccagtcgca gtggctcata cctgtaatcc gagcactttg 18960gtaggctgag gtgggcggat cacttgaggt caggagtttg agaccagcct ggccaacatg 19020gtgaaaccct gtctctacta aaaatacaaa aattagccag gcgtggtggc gggtgcctgt 19080agtcccagct acttgggagg ctgaggcagg agaatcactt gaactgggga ggtggaggtt 19140gcagtgagct gagatcactc cactgcactc cagcctgggt gacagagtga gactccatct 19200taaaaaaaaa aaagactcca tcttaaaaaa aaaaaaaaag aaaaaagaaa gaaaataccc 19260aataagtagt tcctgaatga atagatgaga atgctgttta gaaggttcat gaattggaaa 19320ccgtgattgc tagggaggct ttgagttgat ggtattgtgt tgaaccatgt gttacccagg 19380atcaatttag attttacact ttgttttctc tgttcctttt tatagtaatt ttctgtatgt 19440ggtgttttcc ccccatgaga ttgtatacca tttctcagcg agaactgtgt gtaatgcttg 19500gtggctccct catggtgcct tgcatggaat tggacttcgt ttcagtggat ctgatcccag 19560ttatgttaat gctcgatgga gctaagtctt atctcgaagc agtccatgtc ttcatcagct 19620ggccctgcct ccatgccctg cacagaccat gccactctgg agaggtagtt tccctgtggc 19680ttattagtct tatgttccag tgtgctggcc aagtatgaga gacatcagtg gtatgagaga 19740gtctctctca ttcaaacttc gtaggttttg tagctgggac tgaccagtgc tgacaggaaa 19800tagaggcatt tattaaaagc cagagatttt tcaagttgca ggaagcaaag ctcttgttag 19860ctatgatttt gtggtgggtt tggtagtcca atataaaagt aaaaactgga tgacaatggg 19920aggagcatgc ttgggtctcc aaagttagat catttttcct aagtaatttg tctttaaact 19980tttactggtt tggaatttcc tgagattttg atcttgccag aaagtttata gcaaaagttc 20040tgagcagatg acacttttgc gtctgaaacc aaatcattgt ttttgttttt aacttttttc 20100ttaatatatt atccttagtt cagccctgaa gattattctg ttatttgtgg atctcaactt 20160tccccccatc tcctggatct ttgtgaaatg aatggtatta attgaataga gaaggaagat 20220ataaacataa acttagtcaa aaacttgttc ttgactaggc aagttgggct ttatagcttt 20280gagctgatga catgtctatt cttgtgaaaa agggattttt agtgttggtt tggcttcttg 20340ttatatttga tttattatta ttatcattat cattattttt gagacagagt cttgctctgt 20400cgcccaggct ggagtgcagt ggctcaatct cggctcagtg caacctccgc ctcccaggtt 20460caagcgattc tcgtgcctca gcctctggag tagctgggat tacaggcggg tgccactaca 20520cctggctaat atttgtattt ttagtagaga caggtttcac catgttggct aggctggtct 20580tgaactcctg acctcaggtg atccacctgc cttggcctcc caaagtgctg ggattacagg 20640ccttagccac tgtgcctggc tgattttttt tttttttttt tttttaggtt tgttttaact 20700ggaactttac gtgaatgtaa ttgaatttag aataaaagca cttaatttca cagtgtgcag 20760tgaactttct gttacttatt ttaacagtaa aaccccttgc agtaaatgac ttggagcaaa 20820gattgctttt ttaaaaaatg ttttaatttg tttttctttt cttgagatgg agtcttgctc 20880tgtcaccagg ctggagtatg gtggcgcgat cttggctcac tgcagcctcc ccgcctccta 20940ggttcaagcg aatctcctgc ctcagcctcc tgagtagctg ggactacagg cacatgccac 21000catgcccagc taatttttgt atttttagta gagacagggt ttcaccatgt tggtcaggat 21060ggtcttgatc tcttgacccc gtgatccacc ctcctcggcc tcccaaagtg ctgggattac 21120aactgctggg attacaagtg ctgggattac aagcgtgagc caccacgcct ggccaatttt 21180tttttttttt ttctttttga gacagagttt cactctgtca cccaggctgg agtgcagtgt 21240cacagtcaaa actcactggc agccttaacc tcctgggctc gaatgatcct cctgcctcag 21300cctcccaagt aactgagact acaggcatgt accactgtgc ccagctaatt gtttttttat 21360tttttatttt ttgtagggac agggtctcgc tattttgccc aggctagtct acaactcttg 21420ggctcaagca gtcctcctgc cttgacctcc caaaatgttg ggattacagg gacaagccac 21480tgcacctggc caaggattgt tttttaagtg aactgagacc cagccttatt agtggtccca 21540gagcagacct gggacctgaa gggaaccctt ttcttctggt ccagcgtctt tcctctgatg 21600ggctactttc ctggagcctt tgattgcctg tcatcagagt aactgagttt gaacagagta 21660ggtagttcct ctccagacca ccacactcac cagctttcat tctgcttctc tcgtttagac 21720tgtggttctg aatcctcagt tctatttact gagtgttttt aaacataaaa atgcctttta 21780atgagattga aggccagagg tgggacagtt gaggacaaag tagaaataaa accttcaagg 21840cggggttgtt ggtgggagtc tttttttgtt tgtttgtttt ttgagactga gtctcgctct 21900gtcacccagg ctggagtgca gtggcacaat ctcagctcac tgcaacctcc gcctcccgag 21960ttcaagctat tctcctgcct cagcctcctt agtagctggg atttcaggct cccgccacca 22020tgcccagcta atttttgtat ttttggtaga aacggggttt caccatgttg gccaggctgg 22080tctcaaactc ctgacctcag gtgatctgtc tgcctcagcc tcccaaagtg ctgggattac 22140aggcgtgagc cactgtgcct ggcagggagt cttatagaag ctgtcgtgga caatgtggga 22200agtagtgagc ctttgtattc cagtatgctg ggctccactg tgcttgctct ggcccccggt 22260cgctctctgt gtgttattga gtccccatcc acggccatac tcttcgtcct gcttctctcc 22320ttaccatcct ctccccgcta gtggtaccac ggctaccact agcaattact gacatgtggg 22380atcttagggc tacttcccta taaggctgca gggcatgtgg tgttggctac gcgcatggta 22440accatggtag ccctgtggtt ctccacatgt gcgccttgtg acctgggatt ggctgcagac 22500tagtaataaa ctgcgtcttc tggtatggaa tctgtctgta gttgtacttt ctacctctgt 22560atttaagggg agatctgtaa cctaccaatg ccagttgaag aggatggatg atagagatgt 22620taacaaacag ctgaaaaact aactacaatg gcctgcaaaa tagaacagca ggtttttgtg 22680gcaaaacttt gtgtccatga gtttgttttt taaatatcct catataatct gttttaaatc 22740gagaggcttt gggtaaaagc catggctagt cttacatgtc atggagtacc tagcttgtga 22800ggttcacagt ttattattta cagagtgtcc ccttaaatct tctttgggtc ggttcagcga 22860atgttgctca gatggacttt tttggctgac atagagtcaa aatggtaatc aagcatgaaa 22920gtacagacag tccttaacgc acaaatgtgt catgcttgaa aagttggaaa gttggttctc 22980tggagctctg attgtattgt cctgtagaat ccgtgttgtg aatggtggtt aaatcccaaa 23040tgagtccgta gaacctatat aatctgcaat atacctgcag tattccaatt aatatgtaat 23100tcccccatag aactatgtta atgatttgta tgtatggtat ttaatattat acataataat 23160gattgtatga ataaaaaaca ttctgggctc catgtggatg atggggtgtg tgtgtgtgtc 23220tgtctatgtg tgggtgggtg tgtgttcata gatccctttt cctgcaatcc tggcactgga 23280attggtttta tcatttccaa ttaagtttca ttcccatgaa ttttggagta cagactgggt 23340ccaggtatgc agggcataga ttagagccct gagaaatagg attaggctgg aattgctggg 23400ttggagatca gtagcttcca ggaacacttt ttgggcctgg ctgtcttcat tatccccttt 23460tgttttctcc tggggtctgc aggtattgcc ctgttttgtt cctctaatat cacttttttt 23520ttttttctgc ttttgaccag ggtttttgcc tctggtctac aactgaatat cctatcagac 23580tctcctgatt ttgaaataaa tatatagttt ttttgaggtg ttctagcgaa tttctaaatc 23640taaatgttgt ggcagagtta ttacatacta attttgctat gagaggttgt agaatcccag 23700atgactaatc ttgtaaacca tacacgcatt tccatctaat tctccattgt atatcatgtt 23760gcagaaaata acagcctcta gagtttacat tgcctccttt gactatattt cttatttaag 23820attagttttc agataagacc ttttcatggc agtacataac tgtacagagg gcttccaact 23880tgtcttggga gctctcatct ctgggagaca tcacattacc cactgccccc tgccccccgc 23940ccccagcctg gatgcactca gcctgtaccc catttctgtc ctcagccaaa cactgctgaa 24000atgcaagagc tttcaattgc tagccagtga agatgcagac taagggattt ccatgtagaa 24060gcccgctctt ttcagctggc tcgtcgagag ctggaggccc cttgcttgtt cacatgaggc 24120tttttgtccc tgacttggtg gctgctgttt cacttctcag cagaaaggga cacccttgcc 24180cccccccaga aaggaagatt tgatgtacca cttccgaaag gttcagtcgg gcatcactgt 24240aaccaagaag ataggtcagg tgaggctgga ggtggaacag ggctgctcgc tagaactcca 24300gattgttcca caagtgcctt ctggcagaga atgatggaag cttccgtgat ttttttttct 24360ccttaatagt tatgagcaca gaagaggagc agattgtctg gctatagaag ctgtcttatt 24420ttttattttt gtttttgaga tggagtcttt ctctcttgcc caggctaaag tgcaatggcg 24480cgatctcggc tcactgcaac ctccgcctcc cgagttcaag cgattctcct gcctcagcct 24540cctgagtagc tgggaattac aggcatgcgc caccatgcca gactgatttt tgtattagag 24600acagggtttc accatgttgg tcagtctggt ttcgaactcc tgacctcaag atctgcccac 24660ctcagcctcc caaagtgttg ggattacagg tgttagccac tgcacccggc cgaagctgtc 24720atattaaata gcactttctg cttttagcaa atttaatcca aatgagactt tagattttct 24780tgctctgact taccagcagt tccttgaaac acatttaatt atttttgcca gaaaatcact 24840caagcactta cgccattttt ttaccgtgaa aatatgctgc attattttaa aatatattag 24900aagtcagtaa ccataagatt ttatatgttt tctaatgtat tctgtaagct ttctgctgct 24960tttgtttgga aggtgtattt tgtaacgtag aggactgctt tatctgcttg taagcttgat 25020ttttgttttt actgtaattt ttttttcttt tgctgtattg agaaatacat tgagtaatta 25080taaagtcagt ggcatgttta taagttaata tttgtatcta ttccttagtt actctaactc 25140aaaacctaaa gtaatcttca actctaattt actctgacat ccagttgact gccaagtcct 25200ccaacttaat ccttatcctt ttttttttaa agagatgcag tcttgctttg tcacccaggc 25260tggagtgcag tggtgcaatc atagcttact gtaacctcaa attcctgggc tcaaatgatc 25320ctcccacgtc agcctctgga gtagctgggg ctacaggctc ttgctaccat gcccagctaa 25380ctttttattt ttatttttta tagagacaga gtctcactgt tgctcaggct ggccttgaac 25440tcctgccttc aggcggaact cctgccttca ggcggtcctc ctgcattggc ctcccaaagt 25500gctggaatta caggcccaat tttattcttg ggatgtatgt ctgaaactct ttccttcact 25560tccttcccaa gccttagttc aggcccttct catctgtggt cttcaaagtc gccttcagct 25620ggttcaggtc cttcctttct gctgtatctt tcatgggagg acatgttatg tatcactgtc 25680ctacttgaaa acttccattc cccattgatg agggtgttac ctccagattc ctaacacagg 25740tgctgaaggc atgcctggat aaaggcactc ccttgatctc ctggccaggt ccccgtacac 25800ctgcagcgca tgctccacat tctgtcttta ctgatgctgt gtcttctgcc tgcggagcca 25860cccaccattc tattcacagc ccctgcctca gcggagcacg tgcctccctc ttcctacact 25920gagctgtcct ttctattgaa tcccctcttt tttgtagtat gggaaatatt ttattatgaa 25980tactcttttc tctgttgcct ccgtgaccac gttaactttg ccctaattcg ccttaggact 26040ccatctgctt aggggaaagt taggatttgg ttacagaaag caagctgcta gaaagaacag 26100tgtttagctt ctgacaggca aaataggatt ttgcaacatg cttttccttt ttaatgctta 26160gacattttat atgaattaat atttttattt ggttgcttat acattacttt ctttttagct 26220agaatgtgaa ccctatagga acatggggat tgcctttcac atctttgtat cctcagtacc 26280taatgttcag tcaccctgtg gtcttgtgtc gtatatacat ttagccttcc ttaattaaac 26340catatgtact ggtccccgtc ccccaccccc aaatagagag aaagaaattc cttgaatact 26400acattgccag tatcaaacca caccttgata tcctctgggg aaagggaggt atcagttgaa 26460aagagaaaag aggttaaaat ctaggcatta aaatgtgtaa ggcttagatg ctggcaattt 26520aaggtatgtt ttcctgaggt taattttgat tgtgtgcaaa ttttacctca tatctaactg 26580taggatttag tcaccacata agatgggata cctccataaa tccttcagaa atgtttgtga 26640aattaaataa agccttattg aagactcagc tcttgagagt catctaccta cctaacagtt 26700attcttgaac agaagagtct tacttttccc tataaggcag tgtgatagcc atctgtatat 26760tcatataatt tatgttggcg cttacttcat ttaaaaatgt attccgtgaa tgcagttgcc 26820aggcggtgtg ctgatcagaa acgtgtacca atggcctctt ttataattat aagaggaaga 26880ccaacctgaa acagtcacac aaatgattaa ttttaattgt ggaggagtgc tgggaaagaa 26940aaataaaaga tgcaatgcaa gtgtttacaa aggagctttg agcttgtttg aagtggtcct 27000tgggcactta agcaaggctt aaagaatgat gtgattagaa gtggcttagc aattctaaag 27060aacacaggga aggcgtgtgg ccagaacatt ggtccctaga gcacatcgcc tcctgacata 27120ccatttcctt aagttaatgt tttaccacta tacataggcc ctcccctttg tttacccaga 27180tttttttaat tttaaggatg tttttaataa cttagaatcc tgtaatttgt tgaacagtcc 27240tgtattccct ttacttatat tccttgagat tttataaaat attttttaca tgtcccaagt 27300cttgattata tctttttacc tcttgttaag aaatacttac ttttctattt ttatgctata 27360tttcatgttt actgtagaaa acaaaaaaag taaaattttt ctttattcct atcactgcag 27420cttataagca ctctaaacat tttgatctat attttgccaa tcatatattt tagttaaaat 27480tgttgttgac ataattgtag attcctgtgc agttgaaaga aataatacag agctgagcgc 27540ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcaggcaga tcatgaggtc 27600aggagtttga gaccagactg gccaacatgg cgaaaccctg tctctactaa aaatacaaaa 27660attagctggg tgtggtggcg ggcacctgta atcccagcta gttgggaggc tgaggcagga 27720gaatcgtttg aactccggag gcagaggttg cagtgagccg agatggtacc attccactcc 27780agcctgggca acaagagcaa gactgcatct caaaaataat aataataata ataaataaac 27840tttaaaaata aaacagagag atcccatgtg cgctttgcct agttccccca tccactgccc 27900ataacatttt gcagaactgc agtacagtat cacaaccaca atactgacat tgatacagtc 27960tgctcatctt attcatattt ccccagtgtt actcgtatcc acgtgtgtat gcattgtgtt 28020ttcaatactc ttttattata aagctgtttt taatgtgatt caattctagg ttgttttgtt 28080ctgccctcaa aaagcattcc ctctcctaat catatctccg tcataccctt gtatgttttc 28140tttaaacctg ttttaagaaa gcagctacct gtaagagaaa tgagattgaa aacagaattg 28200ccaatctgct tgtactttat aagcctgttg attgtttaga tacggtttag ccagtttata 28260gttaccctgg gtgctgaaag gtatgctgga tgatacctaa ccaacagaga accattgaat 28320gccgttcaaa atggactgaa gcatcagcaa tgtctgaaaa aggcctgaca gtaatgtaca 28380tgtcaaatgg cccgtaattt aagcagagta gagtaagtag aagaataaac atggggaaag 28440ttccagcaac agaggaggct ttgagctttt gctcttcatc ttgagtggat gttgttctca 28500ggtggtaata ggccatcgag ctttctccac tggctgcctc tctggggaac aaataaccga 28560aaagatactc agcaccctgg ttggtacata ggtggtcagt tgatttatac ttcctggttt 28620tcagtgttgc ttgaattttc taaatggaaa cacagtacct ttataatcag aaaacaatcc 28680cgagttttga tttgagggtg ttgtaaaaag ttaaaaaaaa aaaaacagaa atgtgaaaag 28740gaagttgtgt tagagtattt ggagttgaga aagcatgaaa aggacagaag agaagctggt 28800tgtcaggttg catggggtag ctacaagcac actgaccaga aagtcagctg gaaaaaaaat 28860gtagaaacag gagataaaac ggccaagggg ctatacaagc aaacagcaag gacctgagaa 28920gaaaaactag ttaggtgtga ctgtcagagt gatgtgtaca gtgtgatcct ttctgtgtaa 28980aaacaagcag taagaattcg ctgtttacgt ttgcgtgtgt ttggagaaga gtggggaaga 29040gtaggcactg ccagactgtg aacactggtt aggttattgt tatatctttg tattatatac 29100actggacatg ttatttgtat aatatgagaa gaaattttat aaatcattaa atcttttggc 29160atttaggaac atttgtgttt tctaatagtt gcttctatac tattatcttt attatatgcc 29220cttcatcttc tcagtgtttg gctgttgttg tgattccctt ttgtgagcag tgttgaagtt 29280agctaatatt catttcttct cccttctttc accctcctcc agagtctgat ttgaagtatt 29340cctagctgct acctataaaa gcaataagca agattgtttt acttttcaca aactcgtcct 29400gttctgtgcc tctgcctcgg acatagctgt agtatagagt gttgtctccc ttacatcctt 29460ctatcttaga cctactagta aatattaatg ctcactctaa gttcttctca attctttttt 29520tttttttttt tttttttttt gagaaagagt ttcgctcttg ttgcccaggc tggagtgcaa 29580cggcacgatt tcggctcacc gcaacctcca ccttctgggt ttaagcgact ctcctgcctc 29640agcctcctga gtagctggga ttacagtcac gtgccaccac ccctggcaaa ttttgtattt 29700ttagtagaga caaggtttct tccatgttgg ccaggctggt ctcaaactcc cgacctcagg 29760tgatccacct gcctcagcct tccaaagtgc tgggattcca ggcgtgagcc accgcgccca 29820gcctcttctc tcaattcttc ctgaagctct ttctgcacta gattcctcag gaagggcttg 29880tgggaacaat cttctgtgaa tcaacagtac atattcataa tagtttgtca gcagcctatt 29940attttaaggc catttggtct gtatataaaa atgtttggat cacattttct ttctttaagg 30000taaatatgtt attctgttgt cttctggtat aaagcattgc tgtaaatgtt tgacagtcta 30060attatctttt gcttataagt gacttagggt tttttgtcta tgtgcccaaa ggattttttc 30120cctctttctc tctttttttt tttttttttt tttttttaaa cagacaggat ctcaccctgt 30180tgcccaggct ttagtgcagt gaggcagtca gagcttactg aagttttgaa ctcctgggct 30240tgaggaacaa aggatttttt taacctttta attcaaagtc tcatcattta tgcaaccatg 30300tcttggtgtt ggctgttttg ggttgttctc cctcaaaaat ccatgtgctc tttcaatatg 30360tagttttaaa tctttttttt ttaatttcag gaaaatcttg aattagagtt ttccgttttt 30420cgtctggtac attgcttggg tttccttctt caggaactca gcctgttatg tgtatgtttg 30480atcttctttg cctgtcgtct gtttctttca cttcctctca cttttttaaa cttcatttat 30540taaaaaaaaa tttttttttc gagacagagt ttcgctcttg ttgcccaggc tggagtgcaa 30600tggcgtgatc tcggctcact gcaacctccg cctcccaggt tcaagtgatt ctcctgcctc 30660agtctcccaa gtagctggga ttacaggcat gcgccaccac gcccagctaa ttttttgtat 30720ttttagtaga gacagggttt ctccatgttg gtcaggctgg tcttgaactc ctgacctcgt 30780gatctgcccg cctcagcctc ccaaagtgct gggattacag gcgtgagcca ctgtgcccag 30840ccttattaaa aattttaaaa acatacattt aaacttaaca gaaaaattat gagagagaag 30900ggggtggtgc caggcttttt taaacaacca gctcttacat gaactcatag agtgataact 30960cattaccatg aggacggcat caagccgttc atgaaggatc tggccccgtg acccagacac 31020ctcctactag gtccattttt aacattgggg atcacatttc aacgtgagat ttggaggggg 31080caaaactaca aaccatgtca ctcagggatt ggaggagcaa gtaccaccta tactttggac 31140tcaggtagaa aggcaaaata tccaggaaat aagctgctac cgtccagggt tcagcagagg 31200tgcccatcag cctgccaagt actcaagagt ccagcctcta gggagctaat catcatggtg 31260agctcttcga ggcacaggga gctgggaaga cagtgcttgc cacccctgcc tgaatagtgt 31320ttgcacagag agttctgttg tgtcttgatt gggtcctcct gccactggga atgctgtgga 31380ttatactagg tctctatctg gcttgtttca gggctccatg tgaaaacctt cttgatatcc 31440tagccatcca cctgctcagt ccctagtttg caaggaggct gtggggagcc tagattctgt 31500gtcagataga atgtactaca ttccgtctca ggaatgtacc acatcagaaa acagtgcgac 31560ctgcaggaga agtagaggtg aagaggcaca ttcttccgag aaatgtttct ctcaacaccc 31620agcattccct ggatatcagc aggaaattac tcactgctag aaaatgcccc atgagccttc 31680tgttaaggag gtcaagggag agaacagaga aagttctcaa agttgacttg gtcactggta 31740ctttcttatg cggttcttat tttgtttgcc atcgtcatca tcatgctatg tctattttct 31800caatccaaat ccactgcttt caccttggtt ctttctgacc ggtttggcac actcattcag 31860taaatcctta tggagagccc aatgtctgca taattgtgct gtgctgatga ccaagctaga 31920cctacgagtg tcggctcctt tgagatgtac gggacagctc ttctgtcatc tcttctggga 31980agcctctcca ggcttggtga acagtggcaa gatgtttaac agttgtacat gtgtcccatg 32040ttcctttcta agagcctggg caaaccagac ccggtcgcag gtcatcgtag tatggcgtga 32100gcttcctctc tcctttctga ccttttgtgt gatggcaaga acctgcagag tgacacaagc 32160agcaggcttc tgaggttgct ctagcctcag aatggccgtc ccttctccac cctggccctc 32220attgctgagg tttcctttga agcaacagtg ccggaacaga ctaggggaag cagcttggac 32280atagctgtat gatttattac cacccattga ggccaaccaa agtcggcaag gagaggtagc 32340aggtcagtgg tgcctggaag cttcctcttt cctttgcacc agatgtgact gctctgcaat 32400tactcctaaa tttgctactc tcgtttttac tagccaacct tgatgttttt cccttcttcc 32460tgtagaatag acttcccctc tgatcagtac tttctactca acactatttg tggccacagt 32520gggaactcat tgaggacagg gaccatgaca ttactacctg acccatcaac acttggcata 32580acttgaaatg caaggacaaa aattggctgc aagtacaatg tggtcttcac tctgaaggtg 32640atccttaaaa cttggctttg gcatcatatt gccttaatat acctagggga ttgggtaaaa 32700ccagttactt taaaagagtt ttacaattct ggccttctag ctatcttgtc ttcttaaaca 32760agagcacaag atgaatgtat cttagtgaaa ttttatatgg tttgctttga gtaatcttgc 32820gaagattgat ttttagcaca gtaggaaaga cacattctaa tagtgatttt tttccccgag 32880tttatgtact gctgttgcat gaaaatctga ctagatttaa tgttcctaaa gttctttgtt 32940catcctgatt tttgcaggtc ctagggaaag ctttgttttc ctcttaacct aacttagatg 33000ttgtcatttc atgagctttg gaggaagagt gtatagccaa ttgtgtaatg tctttaaagg 33060atattatctc tgcaatagtt gtttataagg cctaagttat tcatgtaata atagtggccc 33120cggatctgtt tctagcaata ggtatatgga ttttggttcc tatatagttg tagttgtggc 33180tttgagatat tgagcaagcc cttttaagaa aggatttggc atccctcagc cttcaaaagc 33240ttctcaaaat tgatcatatg ttattagcaa aggtttactg cctgcttcca ttgtatagac 33300aatttatttt ttatgtattc cgttctaaga aggcagatga ccaaaagatc ttgcatctgt 33360tgcccaaggc ttgtgactag agaggaaaga gataagaata cttttttaaa atcccatttt 33420actaaatatg ttgaggaagt ggtaagatat attaatttgt tgagattttt ctgttatgcc 33480tattatatga aataggtact ctgaacatgg cttcttaatt aaatatattt gataaaatac 33540aacttgcttc ccctggagtt tagaagtcag ataactgcca tggagagcta tgctttcttt 33600gttttaaaga tctgcttatg aacatgataa acaggaacaa tttaatgttt tcaatatttt 33660cttgtatttt actgcaagtt tatacacaac ataaatatgg gggaaggggg aaatgtttat 33720accagagcca tcctgcccat tctttcctta cagaaggaca aaggagcagt atttatttta 33780actacaaaaa tactattgta ggttttaaaa attccgtata ttttgatatc ttgtgttcct 33840cttgaccttt aatttgctaa atagttgcaa agaatgaagg taacctgcat catcttctta 33900aaaaccaact ctatctaatt ataatagttt gtctatctct gaaaaatagt gatgtgttca 33960ttctgaaatc agaactaccg gatgcagctg cattttgtta ctatttgaat ttcgggagag 34020ggaggaggat gcagcctttc gagctgctga aatacacaaa cacaaagaag acaccaagca 34080tagtagaact gtgttaagct gaccaagcca gaagaagcac ctattctcag catagtatga 34140gacgtaaagg caatataatg ggcatagttg aagatggtag aaggaaaata gactctgatg 34200gtttaatgtt aaatgctttt tttaaaaaag tggtattcca atatcgaaga agaagacttt 34260ctacttttag aagcaataaa ggaaattgca gaggaaaggg tcaataggtt ggaatacata 34320aaaattaaaa acttttaaac tttttttttt tgagacagag tctcactctg tcacccaggc 34380tggagtgcaa tggtgcaatc tcggctcgct acaacctccg cttcctgagt tcaagcaatt 34440ctcctgcctc agcctcccga gtagctggga ttacaggcat gggccaccac tcctggctaa 34500tatttgtatt tttagtagag acagggtttc accatgttgt ccaggctgat ctcaaactcc 34560tgacctcgtg atccgcctgc ctcggcctcc caaagtgctg ggattacagg catgagccac 34620cgcgcctgga ctaaattgtt tcagtattaa ttttttttaa aacaagatct tactgttgcc 34680caggctgaag tacagtggcc caatcatggc taactgcagc cttgacttct gggcctcaag 34740ggatcctccc acctcagcgt cccgagtagc tgggaccaca gacatgtacc accacaccca 34800gctacttgtt ttatttttat ttttgtagag atgaggtttc accatgttgc ccaggctggt 34860ctcgaactcc tgggcccaag caatcctcct cccttggcct cccaaagtgc tggtattaca 34920ggtgtaagcc attgcgccct gcctgatttt ttaaatgtgc aaacagataa gttggaaaag 34980tgatttccaa taaagataaa gagttgatgg ttttaaaata cgtaaagagc ttatatgaat 35040gagaaaaaca ctaacattcc aaaagattag aaggcaaagg acagaaagaa acaaatcact 35100atgtctggga agggacatga aggagcaggt tcccactggg ccagcggggc tcaaacccac 35160tggggacgtc cgagagactg caagggccat gccttcacat tgccgtacct gagaagcaag 35220gagctggggt atttatctct ttcacacttt gggaggctga ggtgggcgga tcacctgagg 35280tcaggagttc gagactagcc tggccaacac agtgaaaccc cgtctctact aaaactagaa 35340ataattagct gggtgtggtg gcacacacct gtaatcccag ctacttggaa ggctgaggca 35400tgagaattgc ttgagcccag gaggtagagg ctgcagtgag cataaattgc accactgcac 35460tccagcctgg gtgaaactct gtctcaaaaa gtaataataa tcatgataaa taaaataaca 35520ttagattgtt agcagaagta gccacaggtt tctcccacct ctctgcaagt tgctgagtgt 35580gattcccatc aagaggtaca atgtcttttt atttttattt tatttatttt atttatattg 35640cctatgttgt ctaggctggt tccaaactcc tgagctcaag tgatccttct acgtcagccc 35700cccaaagtgt tgggattaca ggcatcagcc actgcacctg gcccagatac tttttcttga 35760gtaggaattt cgagtcaccc tgaacattgc atgccttcgt agtggggaag acaataggaa 35820accacaggct gtaggctaaa atgggttgtg tttcttgtaa cgtcatgaca aggcataacc 35880catcttggca tagtaaatag taagcactca ctgaactgat gattttaaat ctttgctgtt 35940tattcagcaa tatcctaaat tagcgctatg ttagtggagt tgcatctccc tcatggatta 36000gtctgaaaaa gatgagaaat ctgtatgtag accaagttat ccttaaactg ctcataatgt 36060atgatgcacg tggttttacg tgtacagcct ggtaccattg ttcttaggca catttcagtg 36120ccagaactct taatacccag gaagaagcaa aaagaaagat ggaggtgcag ctagaggttg 36180tggcctttga acgattcatt ctgccttaat aagagtggtc tggctgagct cggtggctca 36240cacctgtaat cccagcactt tgggaggcca aggcaggcag atcgcttgag cccaggagtt 36300caagaccagc ccaggcagca tagcgagacc ccccctcccc ccgtctctac aaaaaaatag 36360aaacaatgag ccaggcatgg tggaacgtag tgcgtggtgc ctgtagtctc agctacccag 36420ttggctgagg tgggaggatc acctgagccc tagaagtcga ggcttcagtg agcccttatt 36480gtgccactgc actccactct aggtgacaga gcgagacagg tcctgtctcg aaaagaaaga 36540agaagaatta aaaaaagtga ttagatccct tgtgtttggg acacttgttg gcagcaggga 36600tggtagcgtt tatgagggtt gcatgtaaca tcgcctagct cagacatctg tttgactgtc 36660ttcccccctg aagcgcaggc tctgtgaggg caggtctttt gtctttcttg ttaatcttca 36720tatgcttagt gcttgccaca tagttgatgc tcagtcgata tttggatgaa ttgaagggat 36780taatgcattg aatctgaacc ttgctttctt aatgcatatg gggagttctt tggaaagcca 36840cacagaggag cttggttgcc tgcttcctct cttccccaga ttgtcttttt attgttgtgg 36900cttcactgaa gcactctcac ttcaaataat tttgggcatt ggtcgtattt tattctttgt 36960tccttcttca tccttacccc tcagatggta tgtagaaaag tacactacat ctagaaagta 37020ctttataaac tcatttggtt gataataata catatgcctt ttccttggtc ctggtagcag 37080aatcttgtgc cactcttgga atacaaacga aattcttaac caaagccagt ttcattttga 37140tgttctattt tcctcccatt cacactccaa attgtgcacc aaagtatcat cctagttttg 37200tgaggatggt tctccatact tcagggtagg agtatcatgt ggattcctat gatacctttc 37260tccctgggac catggagggc agcagctggt gattgatagt ctgattcccg gtgaggaaag 37320ctgtgagcct tccacttgca gatgtctgcc aactacatgt gtccttagtc aactgtacca 37380ctgtcctccg gcaaacagca gaagcccagg gcctgaagtt cttaagctgt cattatggaa 37440agcagaaggt aaacaaaaca gaagtgaaag tagatttaat tttttagact gttctcttac 37500aggaatggtt ttgtggttct cagcatttta aaaaaaatag tggttccaat atgttttatt 37560gacatcaatt actgtaagtc tgattcattt tctgcctatt gatttctacc caaggtgaaa 37620ttcatgacat ttaacagaaa gcataagtga ttttttaaaa gcagacacta ttagggacgg 37680taaaaataag atttaaagtc gggacacttg aaaaagcaat ttttatacct ttggtaacga 37740ttctattctg attctttgta taaataatat aaacaaaggc tctagaagct tactataatg 37800aagttggtgt gctgtttcta aattctggtt taaggcccaa attcatttta tctgcattaa 37860cttttttttt tttgagagtc tcgctctgtc acccaggcta gagtgcaatg gtatgatctc 37920ggctcactgc aacctctgcc tcccgggttc aagcgattct cctgcctcag cctcccgagt 37980agctgggatt ataggtgtgc gccaccacgc ccggctaatt tttgtatttt tagtagagac 38040ggggtttcac tatgctggtc aggctggtct caaactcctg accttgtgat ccgcctgcct 38100cggcctccca aagtgctggg attacaggcg tgagccactg cacccggccg tgttaaaatt 38160tttcagtggt agaccactat gtcaatatgt tgctttcact gacaacagta ttttcttaaa 38220gataggatac cccatttcta gatgaatctc attctagctg gaaaataatt tttcagttct 38280gaaactacat caggcctcag ggaatcaaaa ctagctatta gccacacaca tataaagtgg 38340ctttgcttta taaacgattt agggtcacca tcaatgacaa tggtcccttt ttattgtatt 38400tttaagagtt tcttatctta aatggctgca taactgtaga gttttaaaaa aattaagtaa 38460atgaccatgt taatgctcta ttaagcttcc aaacaatatt gtaatttact ttgaagattt 38520ttttttattc tcaacatcct gcagcttgac cgtttgcctc cgtgtctcag tgctgcttat 38580tttgaggtgt ggactggagt ccatctgtcc cccttgcctc tgaactgctc cgttttgtgt 38640ttcgtaattc ttcatgctgc atcctgggcg catttctctg tagtagcttt caatttgctc 38700atgctttgac tgggcttagt ctagcgttta tcctatctct taaggttttt taaaaaattt 38760tcatgattat tcatttattt ccaggatttc tcatttcttc agtcacatct ccttgttctg 38820gttttacttc ttcctgtttt tattcataac atctttttta tacacgattc cttcatgtat 38880ttctaatctt aagtatattt aattgcttat ttgattcttt ttttttttta ttgagacagg 38940gtcttactct gccaccaggc cggagtgcag tgacatagtc atagctcact gcagcctcaa 39000ctacttggac tcaagcgacc ttcccacctc agcctcccag gtagctagga atacaggtgt 39060gagagccgcc acacccagct gatttgtctt actatgttgc ccaggctggt cttgaattcc 39120tgggctcatg tgatctgccc ttcttggcct cctgaagtgc tgagattata ggtgtgaacc 39180actgcacctg gccaagtatg tttatttatt tattctaatt tgagagggag tctcgctctg 39240tcgtgcccag gctgtagtgc agtggcacaa tcccagctca ctgcaacctc tgcctcctgg 39300gttcatgcga ttctcttgcc tcagcctcct gagtacctgg ggttacagtt gcgtgccacc 39360acacctagct aatttttgtg tttttagtac aggcggggtt ttaccctgtt ggccaggctg 39420gtcttgaact tgtgacctga agtgatccgc ccgccttggc ctcccaaagt gctgggatta 39480caggcatgag ccaccacgct tggcccaagt atgtttattt ttaaagtccc caacaagcta 39540tacaataaat tgcatatgga atggattttt gttctagttg atttgttggt tatcatttgt 39600agaactaact agttgtcttc tgtgtttgat accttgcttc taggtcattt tgagttggga 39660gccttttgtt ttgtttttat tctcatgctg tttttgagcc tagctgtgcc tttatggttt 39720tctctaaatt taattgacca ttgttttata tttggagcag tgggtgtaca tcagagtgtg 39780aaagcagccc caccctctcc accagaaggt ctccatgcca gtttcacgaa gcatttttca 39840tgccctcatt cctgccctta tcccttgatt tgtggggagt ttgtaaagca gttgattgtt 39900ttttttccac gtagttttcc aagtgcacat aattgttctg ttagtgactt gtagctccat 39960tatctattaa ccttgcccca gaccactgta caagcggacc caacgcttcc tccagctgtg 40020gcagggacag ttacttggta tcctgctgcc ttttcaatgc tgaccagttt tgccccttcc 40080tcccctcaac ccctgtcttt cattcaacta tcaccaaacc aaaagattct ggtttgcttt 40140ttagtatgtg ttcttattca gtacatagtc attttaaaat ttaaaccaaa acagacttgg 40200tactgattag cttaatttta agctttttct ttattattaa acagtgtagt ttatcttagc 40260atttcatatt aagtatatga tttatttcat attgcttata tgaatgtaca cataaatata 40320ataaaaatat tttcctaagg tttttgtagt aaattatatc gtttcattaa ctttcatata 40380tagcattgct tttgacctgg aagacattga acctctgatg atttgtatat tcctcggagt 40440atactttgtt acatagaaat tttctcattt ataatgagat ttgtgattaa caaaatttgt 40500tcaacatgca ttactttgaa gatctggttt ctaaaatttt atgctagtta ccccaccccc 40560ccttctatat atatctccct attcagcgac tactgcaaga gttccaggaa atgtacactg 40620tgtgttcact tactgcattt taaatcattg cctttactat atttctgcat ttcccttcaa 40680tctagctctg tctgtacatt tctgaaagcc agtagcttcc ctgaagaacc aggtaacaac 40740ccgaacaatc aaattagata accatttgta gaatggaggt tccgggagat cttagaagat 40800gtgatgggtg ctaagggact ttgtagttcc ctgaagttcc agtgagtaaa aggtaccctt 40860ggaatttttt attccttcag acttttaaaa cagagatcac tttcaaaaat tactctttct 40920gctttgaatc catgttttag taactatttt gacactgttt ggtcagaagg ctgtgtgggt 40980caactgcaaa taaataaaat aaatgtgatt tcagtaattt ccattttgta acaagtaatt 41040gagaaaatag gattggatca gatatttgct tatacacatt ccctttcagg agcacttctg 41100ttctataaag aatgttggta tattgttaag gacacttcaa gctttgggaa cctttgaagt 41160atccattgat tcagttaaca aaattatgtt gagtgcctac cctgggcctg ggcctgtgtt 41220aggaggggac actaagatga gagtccaaag cacttcttct cagactcctg gctgctaatg 41280ggttgctgcc tctacttctt cacttagcag atagctttaa aatgagtaat gcattttacc 41340atggagcccg taagagacat tcacccagtt gtggaccgag gagaagggtg ttaaacccag 41400attgtgatgt ttcacttgat gaagtgctta atataaacat ggaaatattt ccgcaaggat 41460aaactggctt ttatgcctgt gtgttttcag gagaaataga aatctctaat caaatattgc 41520cagcttttca cccaagtttg actttttgcc taattgagtt tgggaggtgt ctgaataatg 41580gataatgagc tttcctgaat aaatataaaa attaattaac tccaggctct aattcattct 41640gttaccagag ttttgtaagc atgttacccc tttgtgttca ttgggagatc atctgttacc 41700ttcttaaatg agtggggaag gatgggaaat gaggaagagc tataaaaact attcaggtga 41760agaaggtttc tgcccctcct tgcccctttt aaaatctcca gctcagcaga tgctttgttt 41820aaacttgatc aagtgcttgt gaatcttcct agcctagcta aatcataact ttggaaggac 41880ttgctttttt ctctcatgac aatggtttac cacagaaatg attcagatca ctttgtgtgc 41940ctgatgccta tgtaaaatga tacagtgaaa tggaaaccat ttacctgtaa gctttgggca 42000cacccaagcc tgcttcagga gcacatgatc aggcgtgcac tctgggagag ccgtacacat 42060ttgacatcta tgatgtgtgg cgttttattc tatcacattt ctgaaatcta cactaagaga 42120aaggaggctc ttaaaaaacc actgaggtgt ggactggggg aaggagagat ccgtaaagaa 42180cctgtttgtt acctgttgat actatttccc attggtaaaa tttctaattt agtgtgatcc 42240agccctgaaa tgctgaggca cacactgaat gactcctgac atctttagtg tttttgttca 42300ggggactctt ctgggaatct gtttcatggc aagtttatta ttcccttttg gtttggctca 42360tcagtttacc cagcagtcat cttaatcggt tttaaaggct tttattttat tttgttttct 42420ctgtggaaat tttacacatt cagtagatta gaagtagtta tttaatcttt ggttagcata 42480ataaaagatc ttctagggac attttttgct tgcagtggaa ggctagttaa atgtgttcat 42540tagtcatgaa tctgcttttt ctatagctgt tggaaacgta gctcccctgt gatacagttg 42600tagaatacag aaatctcgtt ttgctgttac ggtacggtag tctacttact ttcttccaaa 42660ccattaatgt tatagttacc tttaattgcg taggtcctat cacccctcaa ttttaagact 42720ctaagcctgg cattttatct tacaaaatga aatataaaga cttgtactca gagtatgtgt 42780gtgttttcca tataccattc taaagtagag aaagatgagg gattcgccag aaactgattt 42840ctaataaatt atccagaaac tgaccccttc tcacctcttc tgttactgtc actgtggttt 42900cagccacagc atcctttgct gcattgttac cttagtttcc tgactgtatc cttccttaca 42960ccattgatcc ctgcaatccc atctgcgcgt agcagccaga agggatccac ttactgctgt 43020gatcagaaat cctcagccag gtgcagtggc tcatgcctgt aatctcagca ctatgggagg 43080ctgagactgg agaattattt gagcccagga gtttgagacc agcctcaaac tgggtaatat 43140aatgagacct catctctaca aacaggaaaa aaaaaatttt tttttttttt ttaactagcc 43200aggtatagtg ctaatatacc tgttctggga tccagcatgc tctccctgac ctgcagcttc 43260atctccacca ctttgcccct cactcccacc acaatggctt tcttctcttc ctcagacatg 43320ccgtgcgtcc tcctacctgg aatattcccc tccaaacatt cccatggctc actccctcac 43380cttcatcaga tctctgttcc agtgtcactt ttactggaag gtcttttgtg accatcctac 43440ttattataaa aaaataatct gcccaacctt ctccttttat ttcctctact tgatttttca 43500atttagtact tatcagctga catatatttt gtctctctgt ctctctctgt ctctcataga 43560aggtaaattc tataaaggaa ggaattttta tgtttggttc tttgctgtag ctccaatatt 43620caaaacagtg cctgacacac agtaggccct ttatatttgt tgaataaatg ttgacactct 43680gatatctaat ttttgtctgg tgactaatac gaaaactata gagtgataat aaaagcatta 43740ccttagtaga ctggaaaggg atgagcgcta ggatgaactt tctgcctggc gatcttgctg 43800aatttaggag gcagattggg gttcaaagga ggctgaaatg gctaggattt gcagagcagg 43860gtactaagga tgagcaggct atgacagaaa gaactccaga aatctgcaaa gggatcacct 43920tgagtctggc tggatacagt gtacactttg tagggtgtct cttcatgagc ttggataaag 43980aacaactgtt ggggagtgga taattcccag cactcattca agcttgcatc ggccagaacg 44040gagagagaca gacctctgta atacgtagga tatttggtag aaacattcaa ccgaaaacca 44100tcagatatgc aaaaagtaat aataataagt aaacaatgtg atgcatagct agaagaaaaa 44160tcagacatta gaagcaagcc cagaaatgac agatgataaa ttagcagata aggacattaa 44220aacagctatt ataaataact tagcagattt aaagaaaaac aacataatga ggataatgga 44280agaaaaacaa ccgaatacca tttctaaaga agaaaaatac aatatctgaa atgagaattt 44340agctggatag gattaatagt ttaggcactg cagaagaaaa aaacagcatc tatatgagaa 44400tatacccaag ggaagtacag agaggaaaaa aatgtggatt ggggggtgcc tcagtgacat 44460atggaacaat attaaacaag tctgccccca aaatacttga aggaataagg ttcaagtttt 44520ttccaggttt aatgaaaact ataagcctac agattcaagc atttcaacaa accttcagca 44580aaataaacaa aaccacagta ggcctggcac actgtctcat gcctgcaatc ccagcacttt 44640gggagcctga gtcaggagga ttgcttgaga tctgcttggg caacatagcc agaccctgtc 44700tctacaaaaa ataaaatgaa ataaattagc tggatgtgga ggtccacacc tgtaactcta 44760gctagcctgg aggctaagaa gggaggattg cctgagccca gtagttcaag gctggagtga 44820gctaggactg catcactgca ctccagccta ggcaacagca agaccacatc tctctctctc 44880tctctctctc tctctcaaaa ggcagtgaaa taacgactta tttggggaaa aaataaaggc 44940agagaatttg ttgccagcag actagcataa aaaaaaggaa gtccttgaaa cagaagagaa 45000atgataaaag atggaaattt ggatatatac taaagaatga ggattgctaa aagtgacata 45060catagataaa tatgaaatat atttttattt taaaatttat ttaaagcaaa aataaaaata 45120catcatattt ataacataga aataaaaaat gtatgataat agcataaagg ataagtggac 45180aaatgctgtt gtcgtatttt tggtaaaatg cactattatt tgaaagtaga ccatcgtgaa 45240ttcgatgcat attgtaaacc aaatagaaca ctaaaaaatg aaaataaaga gatatggcta 45300atgtgccaat ggtggagata agatagatgc aaaaaaagaa aaacattcaa aagaaggcag 45360agacagagga aaaaaggacc aaagatcaaa tgagtcaaat agaaagcagc taaactagca 45420atatggcaga tttaaatcta gccatgtcaa tagttatatt aaatgtaaat gttctaaata 45480cctgaattaa aggatgaaga ttgtcagatt agattgaaaa agcatgaccc aactacatgc 45540tgtctgtaag aaattagaaa aagaacaaat taaatccaaa gtaagaagaa aggaaataga 45600gtagaagtta gtgaagtata aaacaaagag caaagaaaat caattaaatg aaaagctggt 45660tctttgtaaa gatcagtaaa attgataaat ttctagctaa actggccaag aaaaaagaaa 45720agacatacaa attaacagta tcaggaagaa aaacagagaa ttcaaaggag tgtaatgcaa 45780actttatgct agtaaatgca ataagttaga tggtatggaa aaaaatgtga acaatacaaa 45840gcagactgtg gttgcctttg gtggcagtag cggggtggga gtggaaggtt gaattgactg 45900gaaccagaag cacaagtgaa ctttttgggg tgatggaaat gttttgtatc ttggttgcat 45960tgatagttaa atggttgtag acattgctta aaactcactg aacacttaag tgggtatgtt 46020ttattatttg taaaatatac ctcaaaagca gttttaaaaa tgtattcaag tacatactta 46080agatctttgc attttactct gagtatacct taattttaaa atctgttttt taaaaagtat 46140tatgtagata ccttttattt tcccaatgtc tttattaaat gacatctcca cgttttgctt 46200cttacctcta tttttttttt tttatttctc tgtctctcag gcatgcacac acacacacca 46260aaaaaagtac atatgcataa tccttttggc tgaataaaat cagttgcaac tgttatttcg 46320gcccttattt gctccgggta aatattcgtt agctgagtgg tttatctgta tcagatattt 46380cttacatctt catccagtca caccagctgg actgaccaga ttgtttttca cttcaagggc 46440agaatttgta ctcactgctg aatgcttcca aatgatacgt agaataacaa atttaagact 46500tagattttta ctttttcagg tctttttttt tttttctgtg ctgtatagca tttccctgaa 46560agcttaatct catctgtaag tgatgcagtg gatgtgttac tattggatta atttatttac 46620tcttaggtag gtttgtaatc tgtcatcatg ctgttgtttt tttgtgtggg tttgtttttg 46680gttttgagac agggtctcac tctgctgccc aggctggaga ggctagagtg cagtgatgtg 46740tttatgggtc actgcagatt caatctcctg ggctcaagtg atcttcctgc ctcaacccct 46800tgtgtagatg gaagcacagg tgcacgccac cacacccggc tattttttta aatgtattgt 46860agagacgagg catcattttt ttgcccaagg ctgatcttga actcctgggc tcaaacaatc 46920ctcccacctc ggctcccaaa gtgctgggat tacagatgtg aaccaccact cgagctccat 46980cattctgtta ttagttgttc tctagtatga gtcaaaaact cttacctgcc cttttacagt 47040tttataaata agtaagcaga atagcagaat gtggacattt tttaaatcca aattgaatat 47100gcacatgact caaggagtca aatagtaccg taatcggttt atgataaaat ccagtggttt 47160ggctgggtgt cgtggctcac acttgtaatc ccagcacctt gggaggctga ggcaggtgga 47220tcacctgaag tcaggagttt gagaccagtc tgacctacat ggtgaaacta ctaaaataca 47280aaattagctg ggcatggtgg tgcatgcctg taatcccagc tacttgggag gctgaggcag 47340gagaattgct tcaacccggg aggcagaggt tgtggtgagc cgatatcgca ttatttcaga 47400acaattttcc acaagatcag tgagtgctgt ccaatagaca tataatacaa cccacataca 47460tgactttaca ttttcttgta gccatagtag aaaaggtcaa aagaagcaga tgaaattaat 47520agcctgggca acaagagcaa aaccccatct tttaaaaaat aaaataaaat atggtggttt 47580gctgtcccca cctcagacca tttctctggt ctttctcatt gaccaccact cccaatcttt 47640gttctgctga ttgattacag cttgtatata tctccatatt tctaagcaaa atgtttatct 47700tttttaaatt tataaattct ttttattatt tttcagagac agggtcttaa ctctgtcgcc 47760caggctggag tacagtggca ccatcgtagc tcactgtagc ctcgaactcc tgggctcaag 47820cagtcttcct gcctccgcct ctcaggtagc tgagactacg ctacaggcac ataccaccat 47880gcccagctca aaatgtttat cttttgatac attattcgag accattatta aggtggatga 47940tttagttttc ttaaacagcc atcccctttc ttttcctccc ctctgcttca ccgcccccat 48000tttcccaatg ttttaccttt tggttaaatc agtactcatt gtttacatta tttgcctctg 48060cacatagtca cagatagtat tgtactgtac tgtactgtgt ttctttttta aacattattt 48120ctgttgttaa taattgactt tttaattttt ttcctatttt gttttttaaa gagatggggt 48180cttactatat tgcccaggct agagttcagt ggctcttcgc gggcatgatc ccactgctga 48240tcagtacagg aatttccacc tgctccattt ccaacctgga ccagttcacc ccttcttagg 48300caacctggtg gtcccccatt cccgggaggt cagcatattg atgccaaact tagtgcggac 48360acccgatcgg cataacgcat gcagcccagg actcctgggc tcaagcagtc ctcccgggct 48420caagcagtcc tcccacctaa gcctcccgcg tagctgagac tacagacact tgccaccaca 48480ccaggttaat ttttgtgttt tttgtagagg tggggttttg ccatgttgtc cagactcatc 48540tcaaacttct cagctcaagt gagcctcctg cctcagcttc ccaagtagct gggattatag 48600acgcatgcca ccacacccca tgataattgc cttttttttt aatttgcata attttctttg 48660tagcttttgc taatgttccc atatcttctt atagccttac agaatgattt tccacaagat 48720cagtgagtgc tgtccagtag acatataata caacccacat acatgatttt accttttttt 48780gtagccatag taaaaaaggt caaaagaagc agatgaaatt aatagtatct tttacttaac 48840ccagttcatt caaaatgtta tttcaataaa tggtcaatat ttaaaatact tgagatattt 48900tgcttttatt tatttctttt gttactaagt cttcaaaatc caatgtgtat tttacactta 48960cagaacatct ctttttagac tggccacatg tagctcaggg ttactgtatt ggacagagtg 49020gtttcagttt caagtttttc cttggagaca tcctacttga aatttccatt ctccatgtat 49080ctgggtggtt ggtctataga cttgccactc acagctgtca tcttgagact ttctttgctt 49140ttcttctcta ttggatattc agtttcctgg atttcaggtc ttctcatttt cctctagtag 49200ttttgttagg tcatggttgg tatggcatgg ttgggatagc gtgttcacac agctatctcg 49260tgagtcatac tcctccaatc cagcctgctc gcttcccgtg tctgtcatgt agttgtcacc 49320ctgctatctc tccctccagt ttttgcagaa atttcctttg tcttcactct tggtcttcct 49380ctcccatccc ccatgtatcc tatatctttc tctttcttgg tttatttcat cactcaggtg 49440gaaaagatgc tccagtggat tactgggaaa agggggagca tggatgataa aggtattgag 49500accttacacg tcagggaatt tttttttttt tttttttttg agacggagtt ttgctcttgt 49560ccaggttgga gtgcagtggc gccaactcgg ctcactgcaa cctccacctc ctgggttcaa 49620gtgattctcc tgcctcagcc tcctgagtag ctgggattac aggtgcccgc caccacgccc 49680agctaatttt ttgtattttt aatagagacg aggtttcact gtgttggcca ggctggtctt 49740gaactcctac ttcaggcaat ccacccacct cggaatgttt ttattgtccc ttctcatttc 49800atgactgctg ggctaggtat agaattccag aatcattgtt cttagaatct cgaaggcatt 49860gcttcattgc tggccagctt tcagtgttct tgcaaagtct gaagctgtgc taatcacctc 49920atcctttgaa agtgaactgt tttttcttcc cagaaactta cagaacattc tctttgtccg 49980cagaattctg ggattgcaat tactgtgcct tagaatgggt ctgtttttat cattatgaag 50040agtactggat gggtcgggag gttttcttga attacttctt gatgttttct ttccttgtat 50100ttttttgttt gctaattttc tatttttttt tcttggttta ctttcttggg cagggggatt 50160tcttctactt atatttgatt cttcagttga gcttgtcatt tttgctatct tgtttttaag 50220tttcgagaga catctttgtt ttatataaca ttctgttctt aatacataga tgcaagatct 50280tttctttctg agtatattaa tatgtatttg aaatctttct attctctgca gtttgtttcc 50340cccaagggtt tttttttttt tctggttttt gttttttgtt tttatgttag agactttcct 50400gttatatctg gtcatcagtg gtacctgcat gtggtggaga gtaggggctt attggagtat 50460gagaaccttg agcaggtgta aggagcctgt caacactgcg ctggcctcag ggcctctagg 50520gaggctgcca gttgtgcatt ctgaggatac cttttggttg tgccttttgt ctggtcagat 50580tatctagaga tgctctgcct cctacctgga ggagaagggt ctagctgcca gcggtgtgag 50640tgtctcttgg ggaaaaggac tcgagttcct ggtgtttggc ttgtgtatgg ccgcttaccc 50700catttttggt ggagcgctca catcttccac tgtgccaaca gtcttgctgc agttcataga 50760ccttctggtt tacatttttc cagaaagtat gtctttagat ttctgcagaa gtctgaggag 50820catggaagga gcttggggaa tgagatggca atccaggtct tcccagatgg ctctaccttt 50880atcccctgca gggaatccca ctcctccttc ctgactggga gcacagccag agccttggga 50940ggaatctgga gtggaaatct cgggcggtct ggctttctta ctgttcactt gtaattttgc 51000tttctcacaa ctgccaacca ctaatcagcc tgatttccag cttccagaat tctattgctg 51060ttgtctgctc tcctattccc accgtagggg atggggctgt cttttttttt ttttttaatt 51120ttggtaaaac atacaaaaca taaagtgttc cattttagcc atttttaggt acacagttca 51180gtggcagtaa gtacattcac gttgtgtgta tttgtttttt tagtaataaa caatataaaa 51240ttttttaagt aataaaacac aaataaaaga ttgtttaatg tgattatcgt ggaattttag 51300gtgtgatcag gagccatggt gtagtcttct gttgaaacag ggtgatagga tttgtttacc 51360acctcctagg aaagcagttg gatagtttgt tggcataaaa gtacatttta tctattttta 51420ataatcgtag ctttatagaa attgcagttg gaactcccag gcctggcatt caaggctctc 51480tgagatctgg gctacccacc catgtcctcc agccgtctgt cgcacctcct actgcccact 51540cactgttcct ggcatgagat gtgatctcca gcccccatgc ctttgctgtg cagggtgttc 51600cagagtgaat tgtccctcct gtctgtctct ctgccctctt cctcgtcttt ccatcttcct 51660gccccacatc actgcctcct acccaaggcc tgtgctcatt cctcctcggt tttcccccat 51720ggcctggtac atacctctga attatcacct tgcatttccc atattgcccg gctctctttg 51780atgtctgttt ctttgctggg tcttcctcag tgtctgacgg tcagttaaat gtctttattc 51840ttttttgtag gatatccgac atgaagccag tgacttccca tgtagagtgg ccaagcttcc 51900taagaacaaa aaccgaaata ggtacagaga cgtcagtccc tgtaagtatc cacgtggccg 51960gtaccagtct tgctcttcct ttgctgcagg cctttttagt caagactcct ttcgcctcag 52020ggtttagtat aataataaat caatgtagca gaggtttatg acgcgattgt ttcctatagt 52080aaaggcatta gagacttata gtaatagctc atttttccac cattatagaa gggctcaggt 52140ttcagtttct ggaaaattca gtgaagttca aagcactttt cttaagcttt gactgttttt 52200gtgatgaatc attttcctac cagctgaagc agagtatagc aggcataata aaaccttttc 52260tggatgactc agcagcagcg tcattagggc atgagcactg tgttccgctg taatgaagcc 52320ccgcacaggc attcggggtg ggcactgtcg tcccctgcgc tgaatatgca aggcagctct 52380gtctggagtc cccaccgcct ccacccccgc caacctcatc atttttctcc ctctttcctg 52440ctgttagttc ttcctaggat tgtcagtgtg cctgctggcc tgtggcagcc ctgtccgcct 52500tctgagtgat tggctgtcag tctgccggta gctgaaaagt aaataactta acatgttaga 52560atttgcataa agtaaggaaa actggagctg agtacaggac ttgaactgcg ccatctcctc 52620taggccacag aggccttttt gacccccttc caggtcttta gacattgtca ggcagtgagg 52680ggtcgtagct gccagtgtct ccatggtagc gtgctctgcc agggatgcag aagattctcc 52740agtcattcct ccagtgggca cttcctgcag gtcctgtgcc catggctggg agtggtggct 52800gtcattgttc tctgccagaa gggttagcag tgcatcctga cctgacttat gtggcgccca 52860gattcctgga aggggtctaa aaatggacct agacttggtg tagaacgtgt gcctcttggc 52920ctgccaccat ggttccctgc ctggttttgt gtgtcagctc tgccgcttaa gaactgagtg 52980gcttcgggca agttgttctc tctcatagga gtgtgtgaag atgaagcaac ataagctgct 53040tagcccagcg cccagtacct cacgcagaca taagtgctca gtaaatgttg tctgtggtgg 53100ggatggttgt caccaacatc tgaagtgcac ttctaggtca tcaggtgaca tgattggcgc 53160caacacatgg tactcttgat ttagcacatc tcagctgagg cacctcattg atatttgttt 53220aaaaacaaaa acaaaaaacc ttggtgattc tgctgtgaag tcctggccag aaacctccag 53280accgctgatc aacacgcaac agaaccatca ccgttcacct ctttgacatg gtgccaggat 53340accctggatc tctagctttt gctatagttg ctctaattag ggaataatct tgtctttaat 53400attcctttgc tacatttttt aacatttctt atctaaatgg ttttatgaat cagttttaca 53460gagaaaaaaa accagtattt aaaatattct tccaggggct ggtccaagta cagtagtgtt 53520tacaactatg tgatcacaac cagttacaga tttctttgtt ccttctccat ccccactgct 53580ttacttgact agccaaaaaa aaaaaaaaaa aaagttattc cagggaaaca attctccaac 53640tttttcactc ccaatctcac tcctcttatc ttcctcccgt actcctatcc tcctcccgta 53700ctcctatcct cctcccctac tcctatcctc cagtagaaac agtcatttgc tgtgaaggtt 53760atgggggaga atgagtcaag gtagaaggtc acctgctgcc cagctcacag tgctgctggt 53820gatgacagca gtccacagtt acaggcactt gctgaacgag gggctctgta tacacctcag 53880ctcattgact cttcccacaa ccctcttgtc acctaccatt tagcaaatga aaaaaccaag 53940gctctgaggt gagttgtttg cccagagtca cccagtgctg tttgaaccca ctcacataac 54000caaccaatac cattatgtaa tttttgaggt cttttatctc tgtgatccac ttaaaaatta 54060tccaagtatc tttatttgta ctaagcctcc ataatgagaa acagtgttcc agatggtggc 54120tagttttcaa agacatctct ctttggaatt cttctttaga acaaaaagcc ccagaccact 54180tatccccatt catatcccct ttggacctag ggagaaggta ctatttatag gtgatcacct 54240gagtttattg tcccttgtgc tgtgccagaa ataaaggtcc ccacctgctc ttattagctc 54300tactaacagg ataaggaaag tggccctcag agagctactg cttttgtgac aaacaaatga 54360tacaagaaaa aaaaagtggc tttttaattt tagtgacctg gggcaggact tccaaatgaa 54420agtttatttc taaaaactaa aaggtaaatt taatatactt tcagtgtttg ggcttaaatt 54480ctctttcaag tgtctttgtg atatgctctg aattttaaaa atttagaatc attgaagttc 54540attatacttg aactttaaaa aaaaaaaaca aaaacctcgt ataaaggtca aggtatgact 54600tcatgctgct gtgtacttag gtcatttaat cttcaaacca ctggatagag gttaggttga 54660agttcgatct taaatcctac ctactgtagc tcattgtacc agcaacagct gtagggacta 54720ggtggaattc atggtgggtt ttgttccctt ttaaagattg aagccaccat attttctgcc 54780ctctaaaagt ttatgtcagc caggcatggg tggctcacac ttgtaatccc agcactttgg 54840ggaggctgag gtgggtggat cacttgaggc caggagttcg agaccagcct ggccaacatg 54900gtgaaacccc atctctacta aaaatagaaa aattaggtga gcatggtggc ctgcgcctgt 54960aatcccagct actcgggagg ctgaggcagg agaaacattt gaatccggga gatggaggct 55020gcagtgagct gagaacatgc cactgcactc cagcctgggt gacagagtga gactcttgac 55080tcaaaaaaaa aagttatgca tcagagaaca gatcctttga tgccctcctc tgccctgaaa 55140ggtttttggg ggagagtaat aagtatcaca acaagatatg acctgagaac agatttccca 55200gataggacat gatccatgtt ttaatatggc ttactgctgt tgcttcatag tgtgaagctt 55260cagacacttc tgaaaaccct ttcagaaaat cccagtcgcc ccatactgat gactaatctc 55320aactaaaaca gggcttcagc cagtgtgaat gccactaatg ccaccaactc acctttgctt 55380ttctgtaggg tgtgcacctg tatgtacaca ttcagctttt ccgggattaa cctctgagtt 55440ctggtttgtc tttcagttga ccatagtcgg attaaactac atcaagaaga taatgactat 55500atcaacgcta gtttgataaa aatggaagaa gcccaaagga gttacattct tacccaggta 55560agcagattgt ctgaattttc tatttaatgt caatttaaga gtttgagagt gctgttatcc 55620acacctcaaa taaaatctgc cacatccttt agaaggtcag gatttcagca taccaaaaag 55680cagcaaggaa gggggaaaaa tcatccttca aaggttcagt ttggttataa ggaacgctaa 55740tcttttctgg gaagcataag atgacattgc tggaaatgag agcttataga aaacaacatt 55800aaaatgccag agttgcctgt gtggtctgtt ggcagagaca gcagagccat ggctggagga 55860gggtctgtac ctgtgttgct tccagaagta tttgtcgtag agcacttgtg atggcaaatc 55920taagaacgtt agcagtagac caggaatctc tgtccagagc cattcagagt agctcagcat 55980ggttctcatt ctttggccag aagaaaggca tcattggatc atgtgaacaa gcatgaaaaa 56040tgacttaaaa tttctgttgg cttttggcat ctttatggaa acaaaatcct gaaagtggtt 56100taataattga gcctcttgta aaacactcag tggcatgtga ccaaaagggt atctgggaaa 56160gaggataaaa agagtttctt tttaattaat cttctcaagt cttaacttgt tacctgtaag 56220ttggtctaaa aagactgggt ttcttatttt gtttttcatc ataatttttg tttctcattc 56280catgtcagct ttcagtctta tatggcttta ggccacaggg cgattttgaa catttgtaat 56340tttgcttaat aattaggaaa ttaaaattct ggggaagaca gaatgctcta tgaagaaagg 56400ctgctttgag caaggagcta ggtcagggcg cgttcaactg aggcctttct tcactgcctt 56460tttgtcttgt cccagttcct ccccatttat gactaaaatc agcccagatg cttctcgtca 56520tctgggatgc agagcatcag cccagctgtg ttcagtccta tggggccatt gagtaagttc 56580ttggtgcatg gatacagggc aggcctttac caggccctga gcccctggtc ctcccagcac 56640ctctggggta tttaggggag gctgatgggg gagggggttg ataaggcggg agatgtctgg 56700ggatgaggtt gaggcaaaag tgacttcttg aggactttgc tttttggaga agtcaaattt 56760cctacttctt gatttcagcc cttcaactct ggtatggagt caggaagccc tttaaatacc 56820tgttgtcggg tgtatcatgt caagtgttgc attagcaaat gaccatgtat ccttgtgcta 56880ctgtcctgcc taccccgcat cctagcgctt ccttgggaca tgagaagctc tgtctggttt 56940gtgaggtggc actggggatg ttgagaaact gtttacacag tttccctttg ccctggggat 57000ttactaaagg agtcgaggca gcctgacccc aaagcatcac ccctggacac tatgaccgaa 57060acatttcccc agtgcccaaa ccaagaacac ccttcccatt tttttttcag tggtgttcat 57120tatgtaataa tacaagtctc tcttctcatt ttttaaaagt cagaagtaca gaagagcaga 57180gaataatgtc caaggggccc tccttcacct cccccgtgca gtgtcagcta agtgtggtgc 57240gtgtccttgc agatcttagg ggattgtgat ccttcagacc attctaaact ggggtggtgc 57300tgggagttag ggaaggcatg aagggagtag tggagagctg cagtgactgg ggtcttcatg 57360ccagggtgga gaatgcaagg cccaggtggc cagccatgtg ccacgggatt tctggctgcc 57420aagagctgtt tatctgttca ctggggaggg aagagttaaa tgtggtctgc ttttctccga 57480gtcccttcag cacagggagt gctgacttgt cttgttcagg tagtaagttc aagatgagct 57540caggaaagaa agtgagagga cactgagggc tagtggttga gccaagtgtg atgggactta 57600aagggagaag atttaaagaa taaggagctt atgggccggg gacggtggct tacgcctgta 57660atcccagcac tttgggaggc tgaagcaggt ggatcacttg ggtcaggagt tcgaggccag 57720cctggccaac atggtgaaac cccgtctcta ctaaaaatac agaaattagc tgggtgtggt 57780agtgtgcacc tgtaatccca gctacttggg aggctgagac aggagaatcg cttgagccca 57840ggaggcagag gttgcagtga gccaggattg cgcccctgta ctccagcctg ggtgatggag 57900cgagactctg cctcaaaaaa aattaaaaaa aaataaagag gttaggtgaa aatagatgag 57960aatggaaacc atgagaagaa gtgatgctgg ccaaggacat gacaggttct gatgtggagg 58020tgataggcaa tgtctcttcc agccactgct aataattgag acaaactcaa ggcattcata 58080ccctgtgtcc agtaaacatc tgtgcccatt gccaggtgag ctggattgaa atgggccagc 58140tgctcagcag acaccctcat gccccagtga ctctgttccc cttgggccac ctcattgacc 58200atttatgttt ctacatctcc taagtttgtt gggccaagga tggaggctgt ctgccgtcag 58260ggtcctcatt gctgatggta ggaatagttg ctgatgtttc attggatgtt gctgtattct 58320agggactgtg ctaagtactt tatagaaatg aacatacttc attttcacag ttttatgaat 58380agggactatt attagtcaag taagcgatgg ggaaactggg gcagggagcg atgaagtgac 58440ttgcgcaagg tcacaagatg atgtgattgg aaccaagaga agtgttgtgg ttggccacgc 58500ccccacactg cctctcatct gcaccaagga gttttgtccc atagcccaag ggccttgggg 58560acgaatctca gtggaggccc ttagcgggcc tgcctgagcc agaaagcaga atcggcattt 58620ttctgtcctt ggttggccca gccctgaact gagatgcgga aatcgccttt cgctgcctgg 58680tagaaaatgg agctgcagtt actgaccacc aggcagagag aggtgggtcc ctgtcccagc 58740ctcagccacc actctgccta agctgtgggg actgagggcg ctgtcgttag ctgactgcag 58800aaggtgagca cacgctgtag catgttatgt ttcagatgtc acatgttgtg ttattgtgtc 58860tttgcagggc cctttgccta acacatgcgg tcacttttgg gagatggtgt gggagcagaa 58920aagcaggggt gtcgtcatgc tcaacagagt gatggagaaa ggttcggtaa gtctcggctt 58980catttgctgt gtatgtgatc atgcatacca ctccatatag ttaccatttt cgtccagatt 59040tttaaattat ttttcttgcc tttgtatttc ctttacgtag tatttttatt taaaaaaatt 59100aaaacagcag catataaatg catgttggtt gtcaaccagt taatgaagtg aataaaaggg 59160aggaggcgga agaactgcac ggacctcttc gcccccgcct tctcctgtgt ggtgcgtgtg 59220gcgctccgcc cacctgtgct gcctgtgcgg ctctcatcac agtgtggagt tgtgtgtgga 59280gttatggaga cctgctttta tcttgaaaag caagttctta gtgcatcttc atggtgtctg 59340attttttggc tggtgagagt gtggctacct ctgcggagct gtgggagcgg ctgactagat 59400gagatttgcc tccattcagt acctagactc ttgccctgcc acacctcttc ggagtgagca 59460ttgacttcag gatgtgtgtc attctaagtt cctgcaactt ttcaaacacc cctcgggcta 59520gcgtgtggct gcacggtgtc catttgtgca ggccaccact cctcttgcat ctgggtctag 59580ccacctctcc ttcttgactt accatagttc attttgtacc atgctttcag aatgagcttt 59640ctcaaatcca agtctcacca cggttcttcc cagctgaaaa cccttgtgcg gttccctttg 59700cctcacagga taatacatgg tgtggcttac ggaaccctgc aggtctggcc ctaggcccct 59760ggacacagac ctctcaccac tcttggaact ttagccagga caaagttttc tgtttttagt 59820ttcttaccat gttctctggg ccgaggagtc ccagtgccca cgttcatccc acttgcaggc 59880acccctggac ggctgccccc agctccccaa ctgcctgcat tctcccctgc cctcctcact 59940ctgttggaat agctgagaat agccgatttc tgggcagccg gcctcctgtg tagactgtcc 60000tgtgtagact gtcctgtgta gattgtctgt gtagactgtc ctgtgtagat tgtctgtgta 60060gactgtcctg tgtagactgt ccatgtaaac tgtcctgtgt agattgtctg tgtagactgt 60120cctgtgtaga ctgtcctgtg tagattgtct gtgtagactg tctctgtaga ccgtcgtgta 60180tagactgtcc tgtgtagact gtctctgtag accgtcgtgt atagactgtc ctgtgtagac 60240tgtctctgta gaccgtcctg tgtagattgt ctgtgtagac catcctgtgt agaccatccc 60300atttagacca tctgcctgtg caggcgcagg ccagtgttca gcagggccac aggctcctcg 60360gcctccctgc cctcgctgct ccccaacact gccaaccctg ctgcggggtc caggaggaga 60420tgggctgagg atcgtggaga ccagcaggag cgtgtggccc aggagcaggg aactgggtgt 60480ccttgggcct tgccaggtcc aggctcagct aggacacggc tctcacagct gtcctggttg 60540cctccggcca cagaagaagg tgagggctcc agagaggcca cctttccaaa aaaagcacag 60600tcatggccct agaatgtaaa aaatccaagt gttaagaagg aacacatcaa aggaaacttc 60660agcagtgaaa acttgaagca ttaaccacga agcctctgcc tccaccacac acaaagaaac 60720ggctttagtt actcgcagaa agtcttcctc ttaggacagc gcgtgtttaa aatcataggg 60780gtttggtttg ttttgttttg gggttggggt tttttggggg ttttttaccc ttgcctactt 60840tttaaaaaat gaaagtgttt atttgcccaa caataacaga cagggagctt gcctaagtgt 60900tctgttgatg atataatgta tcttgtctta gaaaaaaact ttttcagtga aaggtggttt 60960ttaaattttt tcttccctcc ttagtagctt gattagtaaa atgtgaagtt acaaatgtga 61020agcaaacccc cacccttcac cactagtcag caattttgag taaagaaaca aagcatcagg 61080tgctcacagc acacactgtc ttagagggaa ggggaagcct ggtggcctgt ggaagccttc 61140agcatagctc catctgcagg cttctgaccc tcagcactac tgacacttgg gctggatcat 61200tgtctgctag ggatccgggc agggagtggc tgtgctgggc gctgtaggaa gtttagcagc 61260atctctggcc tctatccacc agatgccagt agcaccccct ccccagatgt ggcagtcaga 61320tgtgtttctg tctagactcc agactttgtc caacgtcccc tggtaggcca aattgccccc 61380ggttgagaac caccgctcta gatggtattg agggttggga attttaaatc aagacattta 61440ttcagaaatt accagatata gtagcatttg cttcttattt atttctttgt tgctaagtgt 61500ttggcaaaac ctctttgctg tgagcacaag gtttgcttta gcaattgttg tcacattaca 61560gcaaggagtg gtgtccagcg ctgtagttat gtatttgagc agtgtccagt gctgtagtta 61620tgtgttccag cctcaccagg ccctgtgctt cattgtctcc cactcaagac tgaccacaaa 61680tggcccacag atccactgtg acaacctttc cctttgggtt actgtggtgg catcgagaac 61740atggctggtt ggctttgctg tagtttactg tgataactgt gccagcagtc cctgctttcc 61800tttgttaagt atcccattcc actggaggat tacttgggcg tgcagattgg catgaaaagc 61860aatgtatggt ttgagattgt taaagtttct ttgggatcaa cattttcaat tctgtatcag 61920cattatccct cccagagggc tggctgggag aaatcatgag aagttacagt atcttatttg 61980ctcagctaat ctaattataa atgatccaca cagcttgtgg taaaaccagc ttttggggag 62040ttttcattta atgcatactt gtcttctgat ttccttcctt caccaaatag tgtaggatgc 62100tccctcttat ttttggcaaa catgcctgtt atcttttggg accctgggct tcctggaaac 62160cagttatgca gaagatgatt gtgtgtgtta gactggggtc atccagatgg ctagagttct 62220cactggttct gtttaaggat tgactttaga cacctcagtg taggctgcac catggcgtaa 62280gggttgggat tgttgtttag aagggggaag taagcaaggt gagtttaatt ggccattgca 62340gaatctcacc cgtatctccc tcctgaaatc ctcactaaag ctgccgtttg ctttcaggtg 62400ctttcatgca caagacactg cattttgtat cacagggtcc atataattca tttttctctc 62460gtacttagtt ctctgtgtta agaattactt acttagttct ctgtgttaat aatttttggc 62520gaaaccaaat tacccgtcac agggttactg tagatgtctt tcataggttt tccaaacacc 62580acttgcccac ttgtttggga aggccccaag gactgtttaa catctgcctt catggtggaa 62640acagcaacta tgagagatgc tagcatgttg gcactgccat gttcctctgg taccagccca 62700agataggact caatttgagg cctggtgaag tactgtgttc taataaaaat ccatctactt 62760ttcatggccg tatatatcaa tgtaataggg taactggaaa tgtgatcttg tgccttttaa 62820aaattttgtg tgtttaaaac aaaaatttct attggaaatg acagagcata gcttgttgct 62880gtagacacct gagagtcctt aaaaataaat attgggttat tgacacttag ttgcatgaca 62940gaattcctca cttgtacagt tccaaagtct tagtctttac ccagattaca gagggttatt 63000aagcattagg tttggttttg aaagtgagtg cttgctgtct ggaggtgagc tttaagactc 63060gtctgccctg cttatgagat gaggaagggt ggcctcttcc tcctgcattt ctgttcttcg 63120cttccttctc tgtctgctca ctctgtggaa tgcccacccc agcacgggtg gggtggaacc 63180tgtcagatca gtctcttgtt tctggggtct tgaggcatta taagatctag ttgttagaag 63240tgtgggatta attcatcttt tcacattctt ctaagttcct gcttttagct gccacaccca 63300ctttggctaa gtgggggtct tgccatgtaa ttagcgcctc catgccaagt ggcagaattg 63360cttcaatggt gacagattgt ccccattcaa gagttcactt ttggcaactc atcattgatc 63420caggaaggtg acatggatga aactggctaa gacttcagac aggcttgtgt ccagactctt 63480gagaaagctc tgttggcttc tggtctggca ctgtgaagtt tgctgtgatg ctggcaccac 63540aacctggtgt ttcctaattt gtttctccca cattttgctt tggttttgtc ttttgggcag 63600cttccagctc cagtagagca ggaccaatag gcatttgtgg ttctatattc accctcctca 63660cgtgcttcct ggctcctcat tgcccccaga tgatgccaca ggtccctggg cctgctgcca 63720gtcgtctgtg atctgggcct ctgctggccc cttctccagc tgctcttttc agcctcttat 63780ttgcagtcac tgcctaggaa atcctagtca tccttcaaaa cctgcctctt gcacagagct 63840ttctctgatc tctcttttct gtaaccttgg ctgacctgaa acatttccct cttctgaatt 63900cctgctgcat gtccgtagca tttccccctc agccctcccc catagtccac cttgtcactg 63960ctgggcacag cagtgtcttc tgacagacag ctggccctga agtggttccc ttcacccaca 64020ccatcctttg ccccagagga ggtattgagt gggtcagtgc acgtgaactg ccagtgtcat 64080ttgccaaaga gctgttgaca cacgctgaca tttcttttgc tgaaaatcat aagggctttg 64140agcttccctc tgtccaggca catggtcagg ctgacccggt agctctgccc ctgctgacct 64200gccatttttg tccacaacag ttatccatga gcagaaacat ttgtgtaact gaggcagaaa 64260cttagttcaa gtaaaatgtc actaaattcg agtcagtttt tgtcttagac cctaaatgaa 64320accaaatttt cataaatttt cttgttttaa agaaaaattt aatgagctac atttaaactg 64380agaacatcag atagtgtctg agattatcaa aatagaacat caaaagtatt tttctgaatg 64440aactgaacca aaccagaatg aaagggcaag ccctggggag cctgtctcca agccttctct 64500gaaagggagt ctgtatttgg tgataactgc tcagcctctc caaagggcct cacctgctgt 64560ctctcccagt tttattttta attgcctgtg agttttctgt gcagggtaag gcacctacat 64620tctatgccag cagcctgatc aggtcctggg taatgtttga aatggctaca cagaggagtt 64680tcaaagcctt ttgttcaatc tggcttcacc tcgtagacgg tgagaaagcg tcagagccct 64740gcaggatccc gttgccacgt ttgaccgggg agccgatggg tttggaagtc tgagccctgt 64800ctgcacaacc tgccccggtc agcagcttcg tgcccccacc cccatctccc catgaggcag 64860gcatctgtgc tgaccatggc ttccatgttc agaaaccccc aggcctttga gttatcatga 64920agcttgtggg atgtgctcca agcctcctgc catagaaaaa ctgccatatt gctcacaata 64980attcactatt atttgtttcc ccagttaaaa tgcgcacaat actggccaca aaaagaagaa 65040aaagagatga tctttgaaga cacaaatttg aaattaacat tgatctctga agatatcaag 65100tcatattata cagtgcgaca gctagaattg gaaaacctta cagtgagtat agcacacact 65160tcagcacttc aggcggctac tggttcacat gcctcttcct ttatcccttg ggtgatatta 65220cctaatgtca gtgttcctgg cttttgtata ccccgagcaa gatgtggttt gggcactgtg 65280gtgagcggag cttacttgtg tacctaccaa gtgcccaggg agggtggagg ccacagtgct 65340ctctctgacc tttaacaaca gttaacacca gttcttaggg aaaggagagt ttcttaccca 65400aaagactggt tcctgcttgt gcagctgcag agggactgga gcggcagcct gcaagtccca 65460gtgaagcatg ctgccttctt tgtggtcctc agtcttcgag tctgaagaga gggaagaagg 65520ggtatagggg ctcactccag tttcatagct agtgaaagtt ttctgggcca ggtcttgggt 65580ttttttgttg tgggaagagt ttataacacc agctacttgc ttggtaaaag ttggtcttgg 65640aacatggcaa ggcattgtgg caagcagcac tgccgctgaa cgcgctgctc ctggggcttt 65700ggaataattc ccctggatcc gtaacttggg ggtgttcatg tcattctggg gaacagtgga 65760gggagtgcgc ggcagcacct gggggcacca gtgaagagtg gccagccacc aacctctaga 65820acctaactgg ggtcgaatcc tggccccacc ttactagctc atcacagtgt ctccgtttcc 65880tcttctgtca aactcaggtt ttgcgagggt tctgggaggt cctatacggg aagggttagc 65940agttaccatg ggtgtgtagc acgggcttta tctgaaggga aggtggagcc gtagggagac 66000catgtggagt ggggctccag ggctgtgtgg gtggggaggg atctgcttct gggttacccc 66060atgcctcccc ttctcaagta ctacttttta atcatcatgg ctcctgccat tcatttcata 66120gttgatgtaa gccaggtgcg gtggctcacg tctttaatcc cagcacttgg ggaggctgag 66180gccaggagga tcactcgagg ccaggagttc aagaccagct tgggcaacat agtgagaccc 66240ccgtctctac aaaaaaacaa aaacagttag tcagacatcg tggtgctccc ctatagtcca 66300gctactcagg aggctgaggc aggaggattg cttgtgcccg ggagttcaag gctgcagtga 66360gctatgcttg caccactgca ctctagcctg ggtgacagag caagaccctg tctcaaaaat 66420aaataaataa aaaaaatagt agaagtaaga tctagaatgt agcacaggtt accaggacgt 66480aggcaagggg ttcgggctgc ctggctcttg aggatggtag cagtgcagct gatgtgagtg 66540ctttctgccc tctggtggtg accgcgccgg agtcaccagc cctgccatag ccctgatggg 66600gcagagggtt ctgagtacgg tggatggagg tgctttctgg aagattctca ggagtaacat 66660gggcagtgtg ttggaatgtg ctagaggatt tatgcagtag ccttttaaaa gaatgctttt 66720tagcatttgc aagcctgaca ttaagagtga cttctgggaa actatttgct tgttgaggga 66780aactgaattt caacagagca gaagagctgt gcgctttttg cttggcagag tgaatacagc 66840cagctcagag gttttgatgt taggatctgt ttgctccaac agactttgtt tttaaaaggc 66900ttttctcagc catagctgtc tgttctagca caaggctgga atgagttcct tgtgaaagag 66960gtgagcaggt gtgagggagg gtgtcagtgg gcggtaaccc acaccttcaa ggattaaagg 67020aaaacttgca tttggcatgc ttgcttctta ttcaatttta aaatacattt taacggccgg 67080gcacggtggc taacacctgt aatcccagca ctttgagggg ctgaggtggg tggttcacga 67140ggccaggggt tcaagaccag cctggccaag atggtgaaac cccatctcta ctaaaaatac 67200aaaaaaaaaa aaaattagcc gggcgtggtg gcgggcacct gtaatcccag ctactcggga 67260ggctgaggca gagaattgct tgaacccagg aggcggaggt tgcattgagc cgagatcatg 67320ccactgcatt ccagcctggg cggcagagca agactctgtc tcaaaataat aataataatt 67380ttttaaaaat acattttaag tccttttctt ccccacctgc ctccacccac caaatagaag 67440aggtatttct tcttctttaa tgtcattaag gttatatgga taccattttc tagagaggaa 67500agaatgatgg aattgcctag tgtgagtcta gcaattatcc taacatacac aaatttctcc 67560ttgttctgtg ccaagatact gtatttaata tttaatgaac attaaatatt atttactagt 67620gtatttaatg gctgaggcag ggttaaatat gtattatttt catcccagca gagttggggg 67680aggtcctagt aactatgcca tgagctctgt gagggtgagg tggtgtcttt gcccccgcct 67740ccctggcaca gtgactggca catgattggc atagtgtgga cattcgtcaa gtgaaggaag 67800gcatcatgag cagatctctg gcctgaatcc ttctgccatc agctgctcgc caggtggccc 67860tggcactggg ccacagggaa actctccagg ctggtatggt tcctgtctgt ggctgtcttc 67920ccgggcccat gttaggagac tttcacttcc agagcccttt ccctctcagg gccttgctta 67980ccaagtgact ggttcccatt tactaggagc tcttaggtca ttgaagatgt tgcgtactcc 68040ccccagtgag ggctgccttt tgatcacagc cgccagaagc ctcaaggaag gagcagagct 68100ggaaacagac gccaggccat tgcttctgtt cctctggggc agacccagcc acggaagaga 68160cattctggga caagggctgg ggtccacctt tcaaacgtgt ctgcagcagg ctctcagcat 68220ggactctctg cctccaaaca tccacctcct catcggaaaa tggatgggag tgcctgcctg 68280gagcagctgg tgggagagcg cagcgccagc acgtaggaca cactcggttc atgggctgat 68340gccgttcgca ttgactgcct cttcagctgg gtgttgagcc acaccttgga gtcaccagtc 68400tttggagacc aagtctgcta cttttttctc taaagtgaca atcctctgaa acctccagat 68460catcttgaag cccccgtctg aaagttgccc agagccagtg cctcacctgc tgttccttgt 68520tcactttttc acgggaggcc ttgcagggct ttatgacaag attttatggg tggctgccca 68580gcatcattgt gactcgtgag acagagagaa accagttgta accatgtaga cagtggaagt 68640gatagggaga aaagaggtga ggggactctt caatccgaag ggaaatgaag tctaagcagg 68700cgcaccctgc aggttcagtg tcaagcccag ggcctggccc cagggtgtgg tatttgttga 68760ctgggtgtgt ggaccctggg agaaagtctg agaatgaatg ttcctcttag aggtagagag 68820tggaaggtga ctctgtgtgt acttggaatt agtgatttct gtacagatga ttcttttaga 68880atcatcatga gtatttttct ctttcagacc caagaaactc gagagatctt acatttccac 68940tataccacat ggcctgactt tggagtccct gaatcaccag cctcattctt gaactttctt 69000ttcaaagtcc gagagtcagg gtcactcagc ccggagcacg ggcccgttgt ggtgcactgc 69060agtgcaggca tcggcaggtc tggaaccttc tgtctggctg atacctgcct cttgctggta 69120aggaggccct cgcgggtgcc ctggggagct cctctacctg ctctgctgtg atgttttttc 69180ctaagtagaa actgaagcgc tcctcttcca aaatacagag actcactgtg ttagtctgtt 69240tttgcgttac taataaaggc gtacctgaga ctcggtaatt tgtaaagaaa agaggtttaa 69300ctggctcccg gttctgcagg ctgtacaagc atggcaccag catctgctcg gctcctgggg 69360aggcctcagg gagcttccag tcatggtgga aggtgaaggg gagcaggagc aagagatggg 69420ggaggtccca gactcttaac cagctctctt gtgaatgcat tgcctcaggg agggcaccaa 69480gcctttcatg agggacctgt ccccctgacc cagacacctc ccacccagcc ccacctccaa 69540cactagggat cacatttcag catgagattg ggaggggaca gacatctaac ggtgttatta 69600acgttgccct tgagaattgg acctggctga cttatatctc ctctctggct ttcagatgga 69660caagaggaaa gacccttctt ccgttgatat caagaaagtg ctgttagaaa tgaggaagtt 69720tcggatgggg ctgatccaga cagccgacca gctgcgcttc tcctacctgg ctgtgatcga 69780aggtgccaaa ttcatcatgg gggactcttc cgtgcaggtc agcattgcct ttgtttgaat 69840ccaggtgtga ccattttaac ttttttgtct ttgaaggagg ctgtcagttg taaaagttca 69900aacaccgtct ggtgtcaggg gaaatagcta cccttcatgt ttaaaatagc tagaaagttg 69960tcaaaatgtt caccatgttg cactttgtgc ctttgaagtg ctcacataga gagcattgat 70020aggaagacga gactttattt tcaaaagatt tcatcttcca agtacatggc tgcagccctg 70080agaggccgag agcccctcgc caagccgtca cctctgctca tgcaaaggga tttcctgaca 70140aaccagccga agtgaacact aataggactt cctcttgctg ctctttcaag gatcagtgga 70200aggagctttc ccacgaggac ctggagcccc cacccgagca tatcccccca cctccccggc 70260cacccaaacg aatcctggag ccacacaatg ggaaatgcag ggagttcttc ccaaatcacc 70320agtgggtgaa ggaagagacc caggaggata aagactgccc catcaaggaa gaaaaaggaa 70380gccccttaaa tgccgcaccc tacggcatcg aaaggtaata tgattgggtc ccagcttgtt 70440ggggtgaggg gaaatgactt tctgttctag aaacacacgc tggtactgaa accctgtgga 70500tgcagcctcc tgttggcaag cagcgcttcc gcatccttgg ggaacagggc gcgtggacca 70560cagccactcc actcctggct gctggaggtc cggtattggg cacagggtgg ccgcaggaca 70620tgagccactt ctgtgggctt ctagtgccac cttgtggtgc ttgttggaat gaggggctcg 70680gagccaccga gtagggtttt tctgcccccc ctgacgacag cgccctcccc caggtttccg 70740gacagtcctg aaatgtgatg tccaggcttg agtgccctca gtccccacag tggtcctttg 70800gggaatgtaa ccttttttat gtggtcttga ttaaatccca ttttacttcc ttgcaggtta 70860acaaccatta ttgagtacct attgatatgt gtggtgtact gagttaacta gaacatgtcc 70920cctggtctgt gttctagacc atcttgctgg gaaaaaggca gacccaaagc atattttggt 70980gggggcccat ggacagtgat gtgatagagg tgtccgctga ggtggtcagg gaaggctgct 71040tgcagtaggt ggccgtgcac ggaaagtttg cagaatgagc aggtgttagt tccagctgga 71100gatgactgcc ggctgtgccc ttggtacctg ctttctggag ggaagtttta agacgtgtgc 71160atacttgacc cagcagttgt atacatggag aaatttactt tgcagcaact ctcaaaacaa 71220gcgtgtaaag atgtgtatag gtagttgtgt ttgttgtggc attgtttgta gtagtgaaaa 71280attagagaca ggccaatgat ataaccaggg acctgatcaa ttatgttctc tcccggtgtt 71340gggatattct gtagctctta aagaatgaga tctgggtgta ctgatgtggc cagacattgc 71400aattgcagta catgagaagg caaatcatac agtagtgtgt acaccagtga gtcctccagc 71460cagataaatc ctcacagtga ccagtcgccc aggcaccttg tgaaccctac cctgggtgtg 71520ggtgctatct gaagtacctg ggggaggggg tgacaagtgg acttcaggct gatgtgggcc 71580ctggcctggc cctccctcca agcagagggg gctggctcgc tggaaggtta acatcatcca 71640actctgtcta cacgtggctt gttttttcct agaattcctg ccacaatagc agcatccttg 71700ccattcattt tctccaaagt gagtaaccca tctctgccct ctgattcctc agcatgagtc 71760aagacactga agttagaagt cgggtcgtgg ggggaagtct tcgaggtgcc caggctgcct 71820ccccagccaa aggggagccg tcactgcccg agaaggacga ggaccatgca ctgagttact 71880ggaagccctt cctggtcaac atgtgcgtgg ctacggtcct cacggccggc gcttacctct 71940gctacagggt atgtttccac tgacagacgc gctggcgaga tgctcgtgtg cagagagcac 72000tggccgctag cccgatggta ggattcagtt ctgtggtgca tctgagccag tctcagaaga 72060aacagatcaa aggtttttaa agtctggaac tgtggaaggg ctaacaagag aattaaggat 72120cgatgcactg gggttttaag gagccctctg gtcccaagaa tataagagtc taatctcagg 72180gccttaacct attcaggagt aagtagagaa aatgccaaat acgtctgttt ctctctctct 72240tttttttttt attcctttgt ttttggaaaa aaatagagtt acaacacatt gttgttttta 72300acctttataa aaagcagctt tttgttattt ctggaacaaa aaaaaacaaa gtaggcactt 72360atgaaacttt ctcataccct taggtgatgt aatcagccat ataatttata tttgatttcc 72420cagggaagga atcccaaact tttacgaatg taaactccct tggagaagag ggttaggacg 72480ctgttgcgct caagcccccc tcagctgtgt gcacactgag ccaggacagg gtctttgagc 72540tttcccacta taagaagaac agcaacaaaa ggccgtctag aaaaacagaa cctgcctctg 72600cttctgctca gggtgtcccc gctgggtttc cattgtcctt tctccattgc tccctcctgt 72660gacagccatc ttgctcatgt accagccctc atcaccccat ccccataaat gggtgtcctc 72720gaggcctctg cctgggggtc agaggtcacc acagggtggc cattggcatg tcaacccgct 72780gttaattcag agaagtgggc tccacctcat tgggagaagt gccatttcag cagaaattca 72840cacgttagac gtgtgttgct gttaagtaag gggaagagag aggactagcc tcagagctct 72900ggccatggaa atgacctcct aagacttttt cgtggtttta aatattttac ctctttccag 72960gtggcatctg agtacatcag atggttttgc aaaatgcaaa caattttttc cttggggatg 73020atttttgggg agagggggct actgtaaaaa ataaaaccaa aacccccttt gctccctcgg 73080aggttgaagt tgccgggggg tgtggccggg gtcatgcatg aggcgacagc tctgcaggtg 73140cgggtctggg ctcatctgaa ctgtttggtt tcattccagt tcctgttcaa cagcaacaca 73200tagcctgacc ctcctccact ccacctccac ccactgtccg cctctgcccg cagagcccac 73260gcccgactag caggcatgcc gcggtaggta agggccgccg gaccgcgtag agagccgggc 73320cccggacgga cgttggttct gcactaaaac ccatcttccc cggatgtgtg tctcacccct 73380catcctttta ctttttgccc cttccacttt gagtaccaaa tccacaagcc attttttgag 73440gagagtgaaa gagagtacca tgctggcggc gcagagggaa ggggcctaca cccgtcttgg 73500ggctcgcccc acccagggct ccctcctgga gcatcccagg cgggcggcac gccaacagcc 73560ccccccttga atctgcaggg agcaactctc cactccatat ttatttaaac aattttttcc 73620ccaaaggcat ccatagtgca ctagcatttt cttgaaccaa taatgtatta aaattttttg 73680atgtcagcct tgcatcaagg gctttatcaa aaagtacaat aataaatcct caggtagtac 73740tgggaatgga aggctttgcc atgggcctgc tgcgtcagac cagtactggg aaggaggacg 73800gttgtaagca gttgttattt agtgatattg tgggtaacgt gagaagatag aacaatgcta 73860taatatataa tgaacacgtg ggtatttaat aagaaacatg atgtgagatt actttgtccc 73920gcttattctc ctccctgtta tctgctagat ctagttctca atcactgctc ccccgtgtgt 73980attagaatgc atgtaaggtc ttcttgtgtc ctgatgaaaa atatgtgctt gaaatgagaa 74040actttgatct ctgcttacta atgtgcccca tgtccaagtc caacctgcct gtgcatgacc 74100tgatcattac atggctgtgg ttcctaagcc tgttgctgaa gtcattgtcg ctcagcaata 74160gggtgcagtt ttccaggaat aggcatttgc ctaattcctg gcatgacact ctagtgactt 74220cctggtgagg cccagcctgt cctggtacag cagggtcttg ctgtaactca gacattccaa 74280gggtatggga agccatattc acacctcacg ctctggacat gatttaggga agcagggaca 74340ccccccgccc cccacctttg ggatcagcct ccgccattcc aagtcaacac tcttcttgag 74400cagaccgtga tttggaagag aggcacctgc tggaaaccac acttcttgaa acagcctggg 74460tgacggtcct ttaggcagcc tgccgccgtc tctgtcccgg ttcaccttgc cgagagaggc 74520gcgtctgccc caccctcaaa ccctgtgggg cctgatggtg ctcacgactc ttcctgcaaa 74580gggaactgaa gacctccaca ttaagtggct ttttaacatg aaaaacacgg cagctgtagc 74640tcccgagcta ctctcttgcc agcattttca cattttgcct ttctcgtggt agaagccagt 74700acagagaaat tctgtggtgg gaacattcga ggtgtcaccc tgcagagcta tggtgaggtg 74760tggataaggc ttaggtgcca ggctgtaagc attctgagct gggcttgttg tttttaagtc 74820ctgtatatgt atgtagtagt ttgggtgtgt atatatagta gcatttcaaa atggacgtac 74880tggtttaacc tcctatcctt ggagagcagc tggctctcca ccttgttaca cattatgtta 74940gagaggtagc gagctgctct gctatatgcc ttaagccaat atttactcat caggtcatta 75000ttttttacaa tggccatgga ataaaccatt tttacaaaaa taaaaacaaa aaaagcaagg 75060tgttttggta taataccttt tcaggtgtgt gtggatacgt ggctgcatga ccgggtgggt 75120gggggggagt gtctcagggt cttctgtgac ctcacagaac tgtcagactg tacagttttc 75180caacttgcca tattcatgat gggtttgcat tttagctgca acaataaaat ttttttctaa 75240agaacatgaa tttggggtgc ttcccatttt tttctttgct taatagagct aaaccaggat 75300gagtaactcc tgtttctttc tatccctgct gatgtgaaac agatgttgtc aatcagctgg 75360ggttagagtt ttccacttct aagaattaac ctcagcatcc ctgcattgcc agcaccctca 75420ggctggagcg ctttccttga ctgtgagctt gttgaacacc ttaggcctca gcccatttcc 75480ttcccaaatt gacgctttgc ctgtgtaggg ccctcagata acttaacaaa cttaccagtg 75540ttgtttgaag aacagtgttt tgagttgtaa tctcaaaacc atatccctta cccaattacc 75600tgtaagacac aatggttacc acatctcagt acgtaaagtc cacttgatat agaattgact 75660tagaaataag acagattagt atagtttttc atttgtgtac aaaattaaac aatgtaaatt 75720ccccccaaag tgattttttt gactttttga agtaattttg gacttgcaaa atgttgccaa 75780aatagtacga agagttcccc agtaccctcg aagtttcctc gactgtttca aagctggctg 75840caggcccagg ctcatgagac tgggaagagg acaggctgtg gtcatgtgga cccacaggg 75899244 20 DNA Artificial Sequence Antisense Oligonucleotide 244 gcgctcttagccccgaggcc 20 245 20 DNA Artificial Sequence Antisense Oligonucleotide245 ccagggcggc tgctgcgcct 20 246 20 DNA Artificial Sequence AntisenseOligonucleotide 246 catctccatg acgggccagg 20 247 20 DNA ArtificialSequence Antisense Oligonucleotide 247 ttttccatct ccatgacggg 20 248 20DNA Artificial Sequence Antisense Oligonucleotide 248 actccttttccatctccatg 20 249 20 DNA Artificial Sequence Antisense Oligonucleotide249 ttgtcgatct gctcgaactc 20 250 20 DNA Artificial Sequence AntisenseOligonucleotide 250 gacttgtcga tctgctcgaa 20 251 20 DNA ArtificialSequence Antisense Oligonucleotide 251 gctcccggac ttgtcgatct 20 252 20DNA Artificial Sequence Antisense Oligonucleotide 252 ccagctcccggacttgtcga 20 253 20 DNA Artificial Sequence Antisense Oligonucleotide253 tccactgatc ctgcacggaa 20 254 20 DNA Artificial Sequence AntisenseOligonucleotide 254 ccttccactg atcctgcacg 20 255 20 DNA ArtificialSequence Antisense Oligonucleotide 255 atgcctgcta gtcgggcgtg 20 256 20DNA Artificial Sequence Antisense Oligonucleotide 256 cgggtgtaggccccttccct 20 257 20 DNA Artificial Sequence Antisense Oligonucleotide257 atggagtgga gagttgctcc 20 258 20 DNA Artificial Sequence AntisenseOligonucleotide 258 ttgtactttt tgataaagcc 20 259 20 DNA ArtificialSequence Antisense Oligonucleotide 259 cagtactggt ctgacgcagc 20 260 20DNA Artificial Sequence Antisense Oligonucleotide 260 tctcacgttacccacaatat 20 261 20 DNA Artificial Sequence Antisense Oligonucleotide261 tttcttatta aatacccacg 20 262 20 DNA Artificial Sequence AntisenseOligonucleotide 262 aagtaatctc acatcatgtt 20 263 20 DNA ArtificialSequence Antisense Oligonucleotide 263 ttcagcaaca ggcttaggaa 20 264 20DNA Artificial Sequence Antisense Oligonucleotide 264 gacaatgacttcagcaacag 20 265 20 DNA Artificial Sequence Antisense Oligonucleotide265 tgcctattcc tggaaaactg 20 266 20 DNA Artificial Sequence AntisenseOligonucleotide 266 ggaagtcact agagtgtcat 20 267 20 DNA ArtificialSequence Antisense Oligonucleotide 267 ccaggacagg ctgggcctca 20 268 20DNA Artificial Sequence Antisense Oligonucleotide 268 ctgctgtaccaggacaggct 20 269 20 DNA Artificial Sequence Antisense Oligonucleotide269 tggaatgtct gagttacagc 20 270 20 DNA Artificial Sequence AntisenseOligonucleotide 270 agagtgttga cttggaatgg 20 271 20 DNA ArtificialSequence Antisense Oligonucleotide 271 gctcaagaag agtgttgact 20 272 20DNA Artificial Sequence Antisense Oligonucleotide 272 tgcctctcttccaaatcacg 20 273 20 DNA Artificial Sequence Antisense Oligonucleotide273 tgtttttcat gttaaaaagc 20 274 20 DNA Artificial Sequence AntisenseOligonucleotide 274 tcccaccaca gaatttctct 20 275 20 DNA ArtificialSequence Antisense Oligonucleotide 275 gctctgcagg gtgacacctc 20 276 20DNA Artificial Sequence Antisense Oligonucleotide 276 aggaggttaaaccagtacgt 20 277 20 DNA Artificial Sequence Antisense Oligonucleotide277 ggtggagagc cagctgctct 20 278 20 DNA Artificial Sequence AntisenseOligonucleotide 278 tattggctta aggcatatag 20 279 20 DNA ArtificialSequence Antisense Oligonucleotide 279 gacctgatga gtaaatattg 20 280 20DNA Artificial Sequence Antisense Oligonucleotide 280 ttcttcatgtcaaccggcag 20 281 20 DNA Artificial Sequence Antisense Oligonucleotide281 gccccgaggc ccgctgcaat 20 282 20 DNA Artificial Sequence AntisenseOligonucleotide 282 tagtgaacta ttgttacaac 20 283 20 DNA ArtificialSequence Antisense Oligonucleotide 283 tgctaagcca cttctaatca 20 284 20DNA Artificial Sequence Antisense Oligonucleotide 284 caggattctaagttattaaa 20 285 20 DNA Artificial Sequence Antisense Oligonucleotide285 tgggcaggat ggctctggta 20 286 20 DNA Artificial Sequence AntisenseOligonucleotide 286 tacaatacta tctgtgacta 20 287 20 DNA ArtificialSequence Antisense Oligonucleotide 287 gatacttaca gggactgacg 20 288 20DNA Artificial Sequence Antisense Oligonucleotide 288 aaccctgaggcgaaaggagt 20 289 20 DNA Artificial Sequence Antisense Oligonucleotide289 ccccaggtca ctaaaattaa 20 290 20 DNA Artificial Sequence AntisenseOligonucleotide 290 aaagcaaagg tgagttggtg 20 291 20 DNA ArtificialSequence Antisense Oligonucleotide 291 gctcaattat taaaccactt 20 292 20DNA Artificial Sequence Antisense Oligonucleotide 292 agtcctcaagaagtcacttt 20 293 20 DNA Artificial Sequence Antisense Oligonucleotide293 gaaagcaggg actgctggca 20 294 20 DNA Artificial Sequence AntisenseOligonucleotide 294 aaaactggga gagacagcag 20 295 20 DNA ArtificialSequence Antisense Oligonucleotide 295 acatggaagc catggtcagc 20 296 20DNA Artificial Sequence Antisense Oligonucleotide 296 attgctagactcacactagg 20 297 20 DNA Artificial Sequence Antisense Oligonucleotide297 ggctgtgatc aaaaggcagc 20 298 20 DNA Artificial Sequence AntisenseOligonucleotide 298 cactggctct gggcaacttt 20 299 20 DNA ArtificialSequence Antisense Oligonucleotide 299 gctgggcagc cacccataaa 20 300 20DNA Artificial Sequence Antisense Oligonucleotide 300 agtcccctcacctcttttct 20 301 20 DNA Artificial Sequence Antisense Oligonucleotide301 cctccttacc agcaagaggc 20 302 20 DNA Artificial Sequence AntisenseOligonucleotide 302 tgtattttgg aagaggagcg 20 303 20 DNA ArtificialSequence Antisense Oligonucleotide 303 acagactaac acagtgagtc 20 304 20DNA Artificial Sequence Antisense Oligonucleotide 304 acaaattaccgagtctcagg 20 305 20 DNA Artificial Sequence Antisense Oligonucleotide305 tcatgaaagg cttggtgccc 20 306 20 DNA Artificial Sequence AntisenseOligonucleotide 306 ttggaagatg aaatcttttg 20 307 20 DNA ArtificialSequence Antisense Oligonucleotide 307 agccatgtac ttggaagatg 20 308 20DNA Artificial Sequence Antisense Oligonucleotide 308 cgagcccctcattccaacaa 20 309 20 DNA Artificial Sequence Antisense Oligonucleotide309 cacctcagcg gacacctcta 20 310 20 DNA Artificial Sequence AntisenseOligonucleotide 310 gaaacatacc ctgtagcaga 20 311 20 DNA ArtificialSequence Antisense Oligonucleotide 311 cagagggctc cttaaaaccc 20 312 20DNA Artificial Sequence Antisense Oligonucleotide 312 attcgtaaaagtttgggatt 20 313 20 DNA Artificial Sequence Antisense Oligonucleotide313 ccctcttctc caagggagtt 20 314 20 DNA Artificial Sequence AntisenseOligonucleotide 314 ggaatgaaac caaacagttc 20 315 20 DNA ArtificialSequence Antisense Oligonucleotide 315 aaatggttta ttccatggcc 20 316 20DNA Artificial Sequence Antisense Oligonucleotide 316 aaaaattttattgttgcagc 20 317 20 DNA Artificial Sequence Antisense Oligonucleotide317 ccggtcatgc agccacgtat 20 318 20 DNA Artificial Sequence AntisenseOligonucleotide 318 gttggaaaac tgtacagtct 20 319 20 DNA ArtificialSequence Antisense Oligonucleotide 319 attttattgt tgcagctaaa 20 320 20DNA Artificial Sequence Antisense Oligonucleotide 320 cgcctccttctcggcccact 20 321 20 DNA Artificial Sequence Antisense Oligonucleotide321 gggcggctgc tgcgcctcct 20 322 20 DNA Artificial Sequence AntisenseOligonucleotide 322 gtggatttgg tactcaaagt 20 323 20 DNA ArtificialSequence Antisense Oligonucleotide 323 aaatggcttg tggatttggt 20 324 20DNA Artificial Sequence Antisense Oligonucleotide 324 atggtactctctttcactct 20 325 20 DNA Artificial Sequence Antisense Oligonucleotide325 gccagcatgg tactctcttt 20 326 20 DNA Artificial Sequence AntisenseOligonucleotide 326 gagagttgct ccctgcagat 20 327 20 DNA ArtificialSequence Antisense Oligonucleotide 327 ggagtggaga gttgctccct 20 328 20DNA Artificial Sequence Antisense Oligonucleotide 328 ccttgatgcaaggctgacat 20 329 20 DNA Artificial Sequence Antisense Oligonucleotide329 aaagcccttg atgcaaggct 20 330 20 DNA Artificial Sequence AntisenseOligonucleotide 330 agtactacct gaggatttat 20 331 20 DNA ArtificialSequence Antisense Oligonucleotide 331 ttccattccc agtactacct 20 332 20DNA Artificial Sequence Antisense Oligonucleotide 332 ccatggcaaagccttccatt 20 333 20 DNA Artificial Sequence Antisense Oligonucleotide333 caggcccatg gcaaagcctt 20 334 20 DNA Artificial Sequence AntisenseOligonucleotide 334 caactgctta caaccgtcct 20 335 20 DNA ArtificialSequence Antisense Oligonucleotide 335 ccacgtgttc attatatatt 20 336 20DNA Artificial Sequence Antisense Oligonucleotide 336 ttaaatacccacgtgttcat 20 337 20 DNA Artificial Sequence Antisense Oligonucleotide337 taagcgggac aaagtaatct 20 338 20 DNA Artificial Sequence AntisenseOligonucleotide 338 cagataacag ggaggagaat 20 339 20 DNA ArtificialSequence Antisense Oligonucleotide 339 gagaactaga tctagcagat 20 340 20DNA Artificial Sequence Antisense Oligonucleotide 340 agtgattgagaactagatct 20 341 20 DNA Artificial Sequence Antisense Oligonucleotide341 gacacaagaa gaccttacat 20 342 20 DNA Artificial Sequence AntisenseOligonucleotide 342 ctcatttcaa gcacatattt 20 343 20 DNA ArtificialSequence Antisense Oligonucleotide 343 ggcaggttgg acttggacat 20 344 20DNA Artificial Sequence Antisense Oligonucleotide 344 aaccacagccatgtaatgat 20 345 20 DNA Artificial Sequence Antisense Oligonucleotide345 ttgctgagcg acaatgactt 20 346 20 DNA Artificial Sequence AntisenseOligonucleotide 346 ctggaaaact gcaccctatt 20 347 20 DNA ArtificialSequence Antisense Oligonucleotide 347 gctgggcctc accaggaagt 20 348 20DNA Artificial Sequence Antisense Oligonucleotide 348 ttacagcaagaccctgctgt 20 349 20 DNA Artificial Sequence Antisense Oligonucleotide349 acccttggaa tgtctgagtt 20 350 20 DNA Artificial Sequence AntisenseOligonucleotide 350 ttcccatacc cttggaatgt 20 351 20 DNA ArtificialSequence Antisense Oligonucleotide 351 atatggcttc ccataccctt 20 352 20DNA Artificial Sequence Antisense Oligonucleotide 352 gtgtgaatatggcttcccat 20 353 20 DNA Artificial Sequence Antisense Oligonucleotide353 cctgcttccc taaatcatgt 20 354 20 DNA Artificial Sequence AntisenseOligonucleotide 354 gtgtccctgc ttccctaaat 20 355 20 DNA ArtificialSequence Antisense Oligonucleotide 355 cggaggctga tcccaaaggt 20 356 20DNA Artificial Sequence Antisense Oligonucleotide 356 caggtgcctctcttccaaat 20 357 20 DNA Artificial Sequence Antisense Oligonucleotide357 gtggtttcca gcaggtgcct 20 358 20 DNA Artificial Sequence AntisenseOligonucleotide 358 gctgtttcaa gaagtgtggt 20 359 20 DNA ArtificialSequence Antisense Oligonucleotide 359 ggaccgtcac ccaggctgtt 20 360 20DNA Artificial Sequence Antisense Oligonucleotide 360 caggctgcctaaaggaccgt 20 361 20 DNA Artificial Sequence Antisense Oligonucleotide361 accatcaggc cccacagggt 20 362 20 DNA Artificial Sequence AntisenseOligonucleotide 362 gttccctttg caggaagagt 20 363 20 DNA ArtificialSequence Antisense Oligonucleotide 363 gtggaggtct tcagttccct 20 364 20DNA Artificial Sequence Antisense Oligonucleotide 364 ccacttaatgtggaggtctt 20 365 20 DNA Artificial Sequence Antisense Oligonucleotide365 agctacagct gccgtgtttt 20 366 20 DNA Artificial Sequence AntisenseOligonucleotide 366 ccacgagaaa ggcaaaatgt 20 367 20 DNA ArtificialSequence Antisense Oligonucleotide 367 gaatttctct gtactggctt 20 368 20DNA Artificial Sequence Antisense Oligonucleotide 368 ccacagaatttctctgtact 20 369 20 DNA Artificial Sequence Antisense Oligonucleotide369 gaatgttccc accacagaat 20 370 20 DNA Artificial Sequence AntisenseOligonucleotide 370 gcctggcacc taagccttat 20 371 20 DNA ArtificialSequence Antisense Oligonucleotide 371 atgcttacag cctggcacct 20 372 20DNA Artificial Sequence Antisense Oligonucleotide 372 ctacatacatatacaggact 20 373 20 DNA Artificial Sequence Antisense Oligonucleotide373 tttgaaatgc tactatatat 20 374 20 DNA Artificial Sequence AntisenseOligonucleotide 374 ggataggagg ttaaaccagt 20 375 20 DNA ArtificialSequence Antisense Oligonucleotide 375 gccagctgct ctccaaggat 20 376 20DNA Artificial Sequence Antisense Oligonucleotide 376 ctacctctctaacataatgt 20 377 20 DNA Artificial Sequence Antisense Oligonucleotide377 gctcgctacc tctctaacat 20 378 20 DNA Artificial Sequence AntisenseOligonucleotide 378 aggcatatag cagagcagct 20 379 20 DNA ArtificialSequence Antisense Oligonucleotide 379 gtcaaccggc agccggaact 20 380 20DNA Artificial Sequence Antisense Oligonucleotide 380 cctgcagctaccgccgccct 20 381 20 DNA Artificial Sequence Antisense Oligonucleotide381 cgctgcaatc cccgacccct 20 382 20 DNA Artificial Sequence AntisenseOligonucleotide 382 accaaaacac cttgcttttt 20 383 20 DNA ArtificialSequence Antisense Oligonucleotide 383 gtattatacc aaaacacctt 20 384 20DNA Artificial Sequence Antisense Oligonucleotide 384 cacacacctgaaaaggtatt 20 385 20 DNA Artificial Sequence Antisense Oligonucleotide385 acccggtcat gcagccacgt 20 386 20 DNA Artificial Sequence AntisenseOligonucleotide 386 gtgaggtcac agaagaccct 20 387 20 DNA ArtificialSequence Antisense Oligonucleotide 387 gtacagtctg acagttctgt 20 388 20DNA Artificial Sequence Antisense Oligonucleotide 388 atggcaagttggaaaactgt 20 389 20 DNA Artificial Sequence Antisense Oligonucleotide389 aatgcaaacc catcatgaat 20

1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding PTP1B, wherein said compound specifically hybridizes with and inhibits the expression of PTP1B.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 107, 108, 109, 110, 112, 113, 114, 115, 117, 120, 121, 122, 123, 124, 126, 127, 128, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 168, 169, 170, 171, 172, 173, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 191, 193, 195, 196, 198, 201, 202, 204, 205, 206, 211, 215, 217, 219, 223, 225, 226, 228, 229, 230, 232, 233, 235, 236, 237, 239, 240, 244, 245, 247, 248, 249, 250, 251, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 267, 268, 269, 271, 275, 276, 277, 278, 279, 281, 282, 283, 288, 290, 291, 292, 294, 296, 297, 298, 299, 300, 302, 303, 307, 310, 311, 313, 315, 317, 318, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 340, 341, 342, 343, 344, 345, 347, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 362, 363, 364, 365, 366, 368, 369, 371, 372, 373, 374, 375, 377, 378, 380, 381, 384, 385, 386, 387, 388, or
 389. 4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 12. The composition of claim 11 further comprising a colloidal dispersion system.
 13. The composition of claim 11 wherein the compound is an antisense oligonucleotide.
 14. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding PTP1B.
 15. A method of inhibiting the expression of PTP1B in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of PTP1B is inhibited.
 16. The method of claim 15 wherein the cells or tissues are human cells or tissues.
 17. The method of claim 15 wherein the cells or tissues are rodent cells or tissues.
 18. The method of claim 17 wherein the rodent cells or tissues are mouse cells or tissues.
 19. The method of claim 17 wherein the rodent cells or tissues are rat cells or tissues.
 20. The method of claim 15 wherein the cells or tissues are liver, kidney or adipose cells or tissues.
 21. A method of treating an animal having or suspected of having a disease or condition associated with PTP1B comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of PTP1B is inhibited.
 22. The method of claim 21 wherein the animal is a human.
 23. The method of claim 21 wherein the disease or condition is a metabolic disease or condition.
 24. The method of claim 21 wherein the disease or condition is diabetes.
 25. The method of claim 21 wherein the disease or condition is Type 2 diabetes.
 26. The method of claim 21 wherein the disease or condition is obesity.
 27. The method of claim 21 wherein the disease or condition is a hyperproliferative condition.
 28. The method of claim 27 wherein the hyperproliferative condition is cancer.
 29. A method of decreasing blood glucose levels in an animal comprising administering to said animal the compound of claim
 1. 30. The method of claim 29 wherein the animal is a human or a rodent.
 31. The method of claim 29 wherein the blood glucose levels are plasma glucose levels or serum glucose levels.
 32. The method of claim 29 wherein the animal is a diabetic animal.
 33. A method of preventing or delaying the onset of a disease or condition associated with PTP1B in an animal comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim
 1. 34. The method of claim 33 wherein the animal is a human.
 35. The method of claim 33 wherein the disease or condition is a metabolic disease or condition.
 36. The method of claim 33 wherein the disease or condition is diabetes.
 37. The method of claim 33 wherein the disease or condition is Type 2 diabetes.
 38. The method of claim 33 wherein the disease or condition is obesity.
 39. The method of claim 33 wherein the disease or condition is a hyperproliferative condition.
 40. The method of claim 39 wherein the hyperproliferative condition is cancer.
 41. A method of preventing or delaying the onset of an increase in blood glucose levels in an animal comprising administering to said animal the compound of claim
 1. 42. The method of claim 41 wherein the animal is a primate or a rodent.
 43. The method of claim 41 wherein the animal is obese.
 44. The method of claim 41 wherein the animal is insulin-resistant as compared to a normal animal.
 45. The method of claim 41 wherein the blood glucose levels are plasma glucose levels or serum glucose levels.
 46. The method of claim 41 wherein the animal is a diabetic animal.
 47. The method of claim 16 wherein the human cells are hepatocytes.
 48. A method of improving insulin sensitivity in an animal comprising administering to said animal the compound of claim
 1. 49. The method of claim 48 wherein the animal is a human or rodent.
 50. The method of claim 48 wherein the animal is a diabetic animal.
 51. The method of claim 48 wherein the animal is obese.
 52. The method of claim 48 wherein the animal is hyperinsulinemic.
 53. The method of claim 48 wherein the animal is insulin-resistant.
 54. The compound of claim 1 comprising 12 to 40 nucleobases in length.
 55. The compound of claim 54 comprising 15 to 30 nucleobases in length.
 56. The compound of claim 1 comprising an oligonucleotide.
 57. The compound of claim 56 comprising a DNA oligonucleotide.
 58. The compound of claim 56 comprising an RNA oligonucleotide.
 59. The compound of claim 56 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 60. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding PTP1B said compound specifically hybridizing to and inhibiting the expression of PTP1B.
 61. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding PTP1B said compound specifically hybridizing to and inhibiting the expression of PTP1B.
 62. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding PTP1B said compound specifically hybridizing to and inhibiting the expression of PTP1B.
 63. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding PTP1B said compound specifically hybridizing to and inhibiting the expression of PTP1B. 